Sample records for cortical neurons adjacent

Full Text Available One of the main pathological hallmarks of Alzheimer’s disease (AD is the accumulation of plaques in the cerebral cortex, which may appear either in the neuropil or in direct association with neuronal somata. Since different axonal systems innervate the dendritic (mostly glutamatergic and perisomatic (mostly GABAergic regions of neurons, the accumulation of plaques in the neuropil or associated with the soma might produce different alterations to synaptic circuits. We have used a variety of conventional light, confocal and electron microscopy techniques to study their relationship with neuronal somata in the cerebral cortex from AD patients and APP/PS1 transgenic mice. The main finding was that the membrane surfaces of neurons (mainly pyramidal cells in contact with plaques lack GABAergic perisomatic synapses. Since these perisomatic synapses are thought to exert a strong influence on the output of pyramidal cells, their loss may lead to the hyperactivity of the neurons in contact with plaques. These results suggest that plaques modify circuits in a more selective manner than previously thought.

Full Text Available Neurons of the mammalian cerebral cortex comprise two broad classes: pyramidal neurons, which project to distant targets, and the inhibitory nonpyramidal cells, the cortical interneurons. Pyramidal neurons are generated in the germinal ventricular zone, which lines the lateral ventricles, and migrate along the processes of radial glial cells to their positions in the developing cortex in an `inside-out' sequence. The GABA-containing nonpyramidal cells originate for the most part in the ganglionic eminence, the primordium of the basal ganglia in the ventral telencephalon. These cells follow tangential migratory routes to enter the cortex and are in close association with the corticofugal axonal system. Once they enter the cortex, they move towards the ventricular zone, possibly to obtain positional information, before they migrate radially in the direction of the pial surface to take up their positions in the developing cortex. The mechanisms that guide interneurons throughout these long and complex migratory routes are currently under investigation.

Extrinsic signals controlling generation of neocortical neurons during embryonic life have been difficult to identify. In this study we demonstrate that the dorsal forebrain meninges communicate with the adjacent radial glial endfeet and influence cortical development. We took advantage of Foxc1 mutant mice with defects in forebrain meningeal formation. Foxc1 dosage and loss of meninges correlated with a dramatic reduction in both neuron and intermediate progenitor production and elongation of the neuroepithelium. Several types of experiments demonstrate that retinoic acid (RA) is the key component of this secreted activity. In addition, Rdh10- and Raldh2-expressing cells in the dorsal meninges were either reduced or absent in the Foxc1 mutants, and Rdh10 mutants had a cortical phenotype similar to the Foxc1 null mutants. Lastly, in utero RA treatment rescued the cortical phenotype in Foxc1 mutants. These results establish RA as a potent, meningeal-derived cue required for successful corticogenesis.

A possible mechanism for the synchronization of action potential propagation along a bundle of neurons (ephaptic coupling) is considered. It is shown that this mechanism is similar to the salutatory conduction of the action potential between the nodes of Ranvier in myelinated axons. The proposed model allows us to estimate the scale of the correlation, i.e., the distance between neurons in the nervous tissue, wherein their synchronization becomes possible. The possibility for experimental verification of the proposed model of synchronization is discussed.

Full Text Available The serotonergic pathways originating in the dorsal and median raphe nuclei (DR and MnR, respectively are critically involved in cortical function. Serotonin (5-HT, acting on postsynaptic and presynaptic receptors, is involved in cognition, mood, impulse control and motor functions by 1 modulating the activity of different neuronal types, and 2 varying the release of other neurotransmitters, such as glutamate, GABA, acetylcholine and dopamine. Also, 5-HT seems to play an important role in cortical development. Of all cortical regions, the frontal lobe is the area most enriched in serotonergic axons and 5-HT receptors. 5-HT and selective receptor agonists modulate the excitability of corticalneurons and their discharge rate through the activation of several receptor subtypes, of which the 5-HT1A, 5-HT1B, 5-HT2A and 5-HT3 subtypes play a major role. Little is known, however, on the role of other excitatory receptors moderately expressed in cortical areas, such as 5-HT2C, 5-HT4, 5-HT6 and 5-HT7. In vitro and in vivo studies suggest that 5-HT1A and 5-HT2A receptors are key players and exert opposite effects on the activity of pyramidal neurons in the medial prefrontal cortex (mPFC. The activation of 5-HT1A receptors in mPFC hyperpolarizes pyramidal neurons whereas that of 5-HT2A receptors results in neuronal depolarization, reduction of the afterhyperpolarization and increase of excitatory postsynaptic currents (EPSCs and of discharge rate. 5-HT can also stimulate excitatory (5-HT2A and 5-HT3 and inhibitory (5-HT1A receptors in GABA interneurons to modulate synaptic GABA inputs onto pyramidal neurons. Likewise, the pharmacological manipulation of various 5-HT receptors alters oscillatory activity in PFC, suggesting that 5-HT is also involved in the control of cortical network activity. A better understanding of the actions of 5-HT in PFC may help to develop treatments for mood and cognitive disorders associated with an abnormal function of the

Full Text Available Recent work has shown that functional connectivity among corticalneurons is highly varied, with a small percentage of neurons having many more connections than others. Also, recent theoretical developments now make it possible to quantify how neurons modify information from the connections they receive. Therefore, it is now possible to investigate how information modification, or computation, depends on the number of connections a neuron receives (in-degree or sends out (out-degree. To do this, we recorded the simultaneous spiking activity of hundreds of neurons in cortico-hippocampal slice cultures using a high-density 512-electrode array. This preparation and recording method combination produced large numbers of neurons recorded at temporal and spatial resolutions that are not currently available in any in vivo recording system. We utilized transfer entropy (a well-established method for detecting linear and nonlinear interactions in time series and the partial information decomposition (a powerful, recently developed tool for dissecting multivariate information processing into distinct parts to quantify computation between neurons where information flows converged. We found that computations did not occur equally in all neurons throughout the networks. Surprisingly, neurons that computed large amounts of information tended to receive connections from high out-degree neurons. However, the in-degree of a neuron was not related to the amount of information it computed. To gain insight into these findings, we developed a simple feedforward network model. We found that a degree-modified Hebbian wiring rule best reproduced the pattern of computation and degree correlation results seen in the real data. Interestingly, this rule also maximized signal propagation in the presence of network-wide correlations, suggesting a mechanism by which cortex could deal with common random background input. These are the first results to show that the extent to

The effects of mescaline upon single corticalneurones were studied, using the microiontophoretic technique. Mescaline elicited excitatory and depressant responses similar to those evoked by noradrenaline (NA) and 5-hydroxytryptamine (5-HI). The responses to NA and mescaline were usually in the same direction, the neurone being either excited by both drugs or depressed by both drugs. The correlation between the effects of mescaline and 5-HT, however, was less consistent. The beta-adrenoceptor blocking agent MJ-1999 and the 5-HT antagonist methysergide were both effective in antagonizing mescaline responses.

Subplate (SP) neurons are important for the proper development of thalamocortical innervation. They are necessary for formation of ocular dominance and orientation columns in visual cortex. During the perinatal period, many SP neurons die. The surviving cohort forms interstitial cells in the white matter (WM) and a band of horizontally oriented cells below layer VI (layer VIb, layer VII, or subplate cells). Although the function of embryonic SP neurons has been well established, the functional roles of WM and postnatal SP cells are not known. We used a combination of anatomical, immunohistochemical, and electrophysiological techniques to explore the dendritic morphology, neurotransmitter phenotype, intrinsic electrophysiological, and synaptic input properties of these surviving cells in the rat visual cortex. The density of SP and WM cells significantly decreases during the first month of life. Both populations express neuronal markers and have extensive dendritic arborizations within the SP, WM, and to the overlying visual cortex. Some intrinsic electrophysiological properties of SP and WM cells are similar: each generates high-frequency slowly adapting trains of action potentials in response to a sustained depolarization. However, SP cells exhibit greater frequency-dependent action potential broadening than WM neurons. Both cell types receive predominantly AMPA/kainate receptor-mediated excitatory synaptic input that undergoes paired-pulse facilitation as well as NMDA receptor and GABAergic input. Synaptic inputs to these cells can also undergo long-term synaptic plasticity. Thus, surviving SP and WM cells are functional electrogenic neurons integrated within the postnatal visual cortical circuit.

Full Text Available BACKGROUND: Acetaminophen (AAP is widely prescribed for treatment of mild pain and fever in western countries. It is generally considered a safe drug and the most frequently reported adverse effect associated with acetaminophen is hepatotoxicity, which generally occurs after acute overdose. During AAP overdose, encephalopathy might develop and contribute to morbidity and mortality. Our hypothesis is that AAP causes direct neuronal toxicity contributing to the general AAP toxicity syndrome. METHODOLOGY/PRINCIPAL FINDINGS: We report that AAP causes direct toxicity on rat corticalneurons both in vitro and in vivo as measured by LDH release. We have found that AAP causes concentration-dependent neuronal death in vitro at concentrations (1 and 2 mM that are reached in human plasma during AAP overdose, and that are also reached in the cerebrospinal fluid of rats for 3 hours following i.p injection of AAP doses (250 and 500 mg/kg that are below those required to induce acute hepatic failure in rats. AAP also increases both neuronal cytochrome P450 isoform CYP2E1 enzymatic activity and protein levels as determined by Western blot, leading to neuronal death through mitochondrial-mediated mechanisms that involve cytochrome c release and caspase 3 activation. In addition, in vivo experiments show that i.p. AAP (250 and 500 mg/kg injection induces neuronal death in the rat cortex as measured by TUNEL, validating the in vitro data. CONCLUSIONS/SIGNIFICANCE: The data presented here establish, for the first time, a direct neurotoxic action by AAP both in vivo and in vitro in rats at doses below those required to produce hepatotoxicity and suggest that this neurotoxicity might be involved in the general toxic syndrome observed during patient APP overdose and, possibly, also when AAP doses in the upper dosing schedule are used, especially if other risk factors (moderate drinking, fasting, nutritional impairment are present.

Aim: To observe the effects of stearic acid against oxidative stress in primary cultured corticalneurons. Methods: Corticalneurons were exposed to glutamate,hydrogen peroxide (H202), or NaN3 insult in the presence or absence of stearic acid. Cell viability of corticalneurons was determined by MTT assay and LDH release. Endogenous antioxidant enzymes activity[superoxide dismutases (SOD),glutathione peroxidase (GSH-Px), and catalase (CAT)] and lipid peroxidation in cultured corticalneurons were evaluated using commercial kits. {3-[1(p-chloro-benzyl)-5-(isopropyl)-3-t-butylthiondol-2-yl]-2,2-dimethylpropanoic acid, Na}[MK886; 5 pmol/L; a noncompetitive inhibitor of proliferator-activated receptor(PPAR)α], bisphenol A diglycidyl ether (BADGE; 100 μmol/L; an antagonist of PPARγ), and cycloheximide (CHX; 30 μmol/L, an inhibitor of protein synthesis)were tested for their effects on the neuroprotection afforded by stearic acid.Western blotting was used to determine the PPARγ protein level in corticalneurons.Results: Stearic acid dose-dependently protected corticalneurons against glutamate or H202 injury and increased glutamate uptake in cultured neurons.This protection was concomitant to the inhibition of lipid peroxidation and to the promotion activity of Cu/Zn SOD and CAT in cultured corticalneurons. Its neuroprotective effects were completely blocked by BADGE and CHX. After incubation with H2O2 for 24 h, the expression of the PPARγ protein decreased significantly (P＜0.05), and the inhibitory effect of H2O2 on the expression of PPARγ can be attenuated by stearic acid. Conclusion: Stearic acid can protect corticalneurons against oxidative stress by boosting the internal antioxidant enzymes.Its neuroprotective effect may be mainly mediated by the activation of PPARγ and new protein synthesis in corticalneurons.

AIM: The neurotoxicity of glutamate is associated with neurological disorders including hypoxic-ischaemic brain injury. Studies using cultured corticalneurons have demonstrated that exposure to glutamate produced delayed degeneration of mature neurons. Oxygen free radicals generated during injury have been postulated to be a major cause of neuronal cell

Full Text Available Neurons may serve different functions over the course of an organism’s life. Recent evidence suggests that cortical subplate neurons including those that reside in the white matter may perform longitudinal multi-tasking at different stages of development. These cells play a key role in early cortical development in coordinating thalamocortical reciprocal innervation. At later stages of development, they become integrated within the cortical microcircuitry. This type of longitudinal multi-tasking can enhance the capacity for information processing by populations of cells serving different functions over the lifespan. Subplate cells are initially derived when cells from the ventricular zone underlying the cortex migrate to the cortical preplate that is subsequently split by the differentiating neurons of the cortical plate with some neurons locating in the marginal zone and others settling below in the subplate (SP. While the cortical plate neurons form most of the cortical layers (layers 2-6, the marginal zone neurons form layer 1 and the SP neurons become interstitial cells of the white matter as well as forming a compact sublayer along the bottom of layer 6. After serving as transient innervation targets for thalamocortical axons, most of these cells die and layer 4 neurons become innervated by thalamic axons. However, 10-20% survives, remaining into adulthood along the bottom of layer 6 and as a scattered population of interstitial neurons in the white matter. Surviving subplate cells’ axons project throughout the overlying laminae, reaching layer 1 and issuing axon collaterals within white matter and in lower layer 6. This suggests that they participate in local synaptic networks, as well. Moreover, they receive excitatory and inhibitory synaptic inputs, potentially monitoring outputs from axon collaterals of cortical efferents, from cortical afferents and/or from each other. We explore our understanding of the functional connectivity of

Acidosis impairs brain functions. Neuron-specific mechanisms underlying acidosis-induced brain dysfunction remain elusive. We studied the sensitivity of cortical GABAergic neurons and glutamatergic neurons to acidosis by whole-cell recording in brain slices. The acidification to the neurons was induced by perfusing artificial cerebral spinal fluid with lower pH. This acidification impairs excitability and synaptic transmission in the glutamatergic and GABAergic neurons. Acidosis impairs spiking capacity in the GABAergic neurons more than in the glutamatergic neurons. Acidosis also strengthens glutamatergic synaptic transmission and attenuates GABAergic synaptic transmission on the GABAergic neurons more than the glutamatergic neurons, which results in the functional impairment of these GABAergic neurons. This acidosis-induced dysfunction predominantly in the cortical GABAergic neurons drives the homeostasis of neuronal networks toward overexcitation and exacerbates neuronal impairment.

Normalization, which divisively scales neuronal responses to multiple stimuli, is thought to underlie many sensory, motor, and cognitive processes. In every study where it has been investigated, neurons measured in the same brain area under identical conditions exhibit a range of normalization, ranging from suppression by nonpreferred stimuli (strong normalization) to additive responses to combinations of stimuli (no normalization). Normalization has been hypothesized to arise from interactions between neuronal populations, either in the same or different brain areas, but current models of normalization are not mechanistic and focus on trial-averaged responses. To gain insight into the mechanisms underlying normalization, we examined interactions between neurons that exhibit different degrees of normalization. We recorded from multiple neurons in three cortical areas while rhesus monkeys viewed superimposed drifting gratings. We found that neurons showing strong normalization shared less trial-to-trial variability with other neurons in the same cortical area and more variability with neurons in other cortical areas than did units with weak normalization. Furthermore, the cortical organization of normalization was not random: neurons recorded on nearby electrodes tended to exhibit similar amounts of normalization. Together, our results suggest that normalization reflects a neuron's role in its local network and that modulatory factors like normalization share the topographic organization typical of sensory tuning properties.

There is growing interest in engineering nerve cells in vitro to control architecture and connectivity of cultured neuronal networks or to build neuronal networks with predictable computational function. Pattern technologies, such as micro-contact printing, have been developed to design ordered neuronal networks. However, electrophysiological characteristics of the single patterned neuron haven’t been reported. Here, micro-contact printing, using polyolefine polymer (POP) stamps with high resolution, was employed to grow corticalneurons in a designed structure. The results demonstrated that the morphology of patterned neurons was well constrained, and the number of dendrites was decreased to be about 2. Our electrophysiological results showed that alterations of dendritic morphology affected firing patterns of neurons and neural excitability. When stimulated by current, though both patterned and un-patterned neurons presented regular spiking, the dynamics and strength of the response were different. The un-patterned neurons exhibited a monotonically increasing firing frequency in response to injected current, while the patterned neurons first exhibited frequency increase and then a slow decrease. Our findings indicate that the decrease in dendritic complexity of corticalneurons will influence their electrophysiological characteristics and alter their information processing activity, which could be considered when designing neuronal circuitries.

This study examined the effect of tonic contraction of the finger muscle on the motor cortical representation of the contracting adjacent muscle. A representation map of the motor evoked potential (MEP) in the first dorsal interosseous (FDI) and abductor digiti minimi (ADM) muscles was obtained with the subject at rest or during tonic contraction of the ADM muscle while the FDI muscle was tonically contracted. The center of gravity (COG) of the MEP map in the FDI muscle shifted medially during contraction of the ADM muscle. Motor cortical excitability in the motor cortical representation of the FDI muscle that did not overlap with the motor cortical representation of the ADM muscle was suppressed, but motor cortical excitability in the motor cortical representation of the FDI muscle overlapping with the motor cortical representation of the ADM muscle was not suppressed during contraction of the ADM muscle. The motor cortical representation of the FDI muscle not overlapping with the motor cortical representation of the ADM muscle was located lateral to that of the FDI muscle that did overlap with the motor cortical representation of the ADM muscle. Medial shift of the COG of the motor cortical representation of the contracting finger muscle induced by tonic contraction of the adjacent finger muscle must be due to suppression of motor cortical excitability in the lateral part of the representation, which is not shared by the adjacent representation.

Full Text Available Neuronal migration in the cortex is controlled by the paracrine action of the classical neurotransmitters glutamate and GABA. Glutamate controls radial migration of pyramidal neurons by acting primarily on NMDA receptors and regulates tangential migration of inhibitory interneurons by activating non-NMDA and NMDA receptors. GABA, acting on ionotropic GABAA-rho and GABAA receptors, has a dichotomic action on radially migrating neurons by acting as a GO signal in lower layers and as a STOP signal in upper cortical plate (CP, respectively. Metabotropic GABAB receptors promote radial migration into the CP and tangential migration of interneurons. Besides GABA, the endogenous GABAergic agonist taurine is a relevant agonist controlling radial migration. To a smaller extent glycine receptor activation can also influence radial and tangential migration. Activation of glutamate and GABA receptors causes increases in intracellular Ca2+ transients, which promote neuronal migration by acting on the cytoskeleton. Pharmacological or genetic manipulation of glutamate or GABA receptors during early corticogenesis induce heterotopic cell clusters in upper layers and loss of cortical lamination, i.e. neuronal migration disorders which can be associated with neurological or neuropsychiatric diseases. The pivotal role of NMDA and ionotropic GABA receptors in corticalneuronal migration is of major clinical relevance, since a number of drugs acting on these receptors (e.g. anti-epileptics, anesthetics, alcohol may disturb the normal migration pattern when present during early corticogenesis.

The density of cells and neurons in the neocortex of many mammals varies across cortical areas and regions. This variability is, perhaps, most pronounced in primates. Nonuniformity in the composition of cortex suggests regions of the cortex have different specializations. Specifically, regions with densely packed neurons contain smaller neurons that are activated by relatively few inputs, thereby preserving information, whereas regions that are less densely packed have larger neurons that have more integrative functions. Here we present the numbers of cells and neurons for 742 discrete locations across the neocortex in a chimpanzee. Using isotropic fractionation and flow fractionation methods for cell and neuron counts, we estimate that neocortex of one hemisphere contains 9.5 billion cells and 3.7 billion neurons. Primary visual cortex occupies 35 cm(2) of surface, 10% of the total, and contains 737 million densely packed neurons, 20% of the total neurons contained within the hemisphere. Other areas of high neuron packing include secondary visual areas, somatosensory cortex, and prefrontal granular cortex. Areas of low levels of neuron packing density include motor and premotor cortex. These values reflect those obtained from more limited samples of cortex in humans and other primates.

The performance of complex networks, like the brain, depends on how effectively their elements communicate. Despite the importance of communication, it is virtually unknown how information is transferred in local cortical networks, consisting of hundreds of closely spaced neurons. To address this, it is important to record simultaneously from hundreds of neurons at a spacing that matches typical axonal connection distances, and at a temporal resolution that matches synaptic delays. We used a 512-electrode array (60 μm spacing) to record spontaneous activity at 20 kHz from up to 500 neurons simultaneously in slice cultures of mouse somatosensory cortex for 1 h at a time. We applied a previously validated version of transfer entropy to quantify information transfer. Similar to in vivo reports, we found an approximately lognormal distribution of firing rates. Pairwise information transfer strengths also were nearly lognormally distributed, similar to reports of synaptic strengths. Some neurons transferred and received much more information than others, which is consistent with previous predictions. Neurons with the highest outgoing and incoming information transfer were more strongly connected to each other than chance, thus forming a “rich club.” We found similar results in networks recorded in vivo from rodent cortex, suggesting the generality of these findings. A rich-club structure has been found previously in large-scale human brain networks and is thought to facilitate communication between cortical regions. The discovery of a small, but information-rich, subset of neurons within cortical regions suggests that this population will play a vital role in communication, learning, and memory. SIGNIFICANCE STATEMENT Many studies have focused on communication networks between cortical brain regions. In contrast, very few studies have examined communication networks within a cortical region. This is the first study to combine such a large number of neurons (several

Golgi-impregnated neurons from rat perirhinal cortex (PR) were classified into one of 15 distinct morphological categories (N = 6,891). The frequency of neurons in each cell class was determined as a function of the layer of PR and the age of the animal, which ranged from postnatal day 0 (P0) to young adulthood (P45). The developmental appearance of Golgi-impregnated neurons conformed to the expected "inside-out" pattern of development, meaning that cells populated in deep before superficial layers of PR. The relative frequencies of different cell types changed during the first 2 weeks of postnatal development. The largest cells, which were pyramidal and spiny multipolar neurons, appeared earliest. Aspiny stellate neurons were the last to appear. The total number of Golgi-impregnated neurons peaked at P10-12, corresponding to the time of eye-opening. This early increase in the number of impregnated neurons parallels observations in other cortical areas. The relative frequency of the 15 cell types remained constant between P14 to P45. The proportion of pyramidal neurons in PR ( approximately 50%) was much smaller than is typical of neocortex ( approximately 70%). A correspondingly larger proportion of PR neurons were nonpyramidal cells that are less common in neocortex. The relative frequency distribution of cell types creates an overall impression of considerable morphological diversity, which is arguably related to the particular manner in which this periallocortical brain region processes and stores information.

Full Text Available While larger brains possess concertedly larger cerebral cortices and cerebella, the relative size of the cerebral cortex increases with brain size, but relative cerebellar size does not. In the absence of data on numbers of neurons in these structures, this discrepancy has been used to dispute the hypothesis that the cerebral cortex and cerebellum function and have evolved in concert and to support a trend towards neocorticalization in evolution. However, the rationale for interpreting changes in absolute and relative size of the cerebral cortex and cerebellum relies on the assumption that they reflect absolute and relative numbers of neurons in these structures across all species – an assumption that our recent studies have shown to be flawed. Here I show for the first time that the numbers of neurons in the cerebral cortex and cerebellum are directly correlated across 19 mammalian species of 4 different orders, including humans, and increase concertedly in a similar fashion both within and across the orders Eulipotyphla (Insectivora, Rodentia, Scandentia and Primata, such that on average a ratio of 3.6 neurons in the cerebellum to every neuron in the cerebral cortex is maintained across species. This coordinated scaling of cortical and cerebellar numbers of neurons provides direct evidence in favor of concerted function, scaling and evolution of these brain structures, and suggests that the common notion that equates cognitive advancement with neocortical expansion should be revisited to consider in its stead the coordinated scaling of neocortex and cerebellum as a functional ensemble.

Full Text Available Broadband spontaneous macroscopic neural oscillations are rhythmic cortical firing which was extensively examined during the last century, however, their possible origination is still controversial. In this work we show how macroscopic oscillations emerge in solely excitatory random networks and without topological constraints. We experimentally and theoretically show that these oscillations stem from the counterintuitive underlying mechanism - the intrinsic stochastic neuronal response failures. These neuronal response failures, which are characterized by short-term memory, lead to cooperation among neurons, resulting in sub- or several- Hertz macroscopic oscillations which coexist with high frequency gamma oscillations. A quantitative interplay between the statistical network properties and the emerging oscillations is supported by simulations of large networks based on single-neuron in-vitro experiments and a Langevin equation describing the network dynamics. Results call for the examination of these oscillations in the presence of inhibition and external drives.

The longer we are awake, the deeper is our subsequent sleep. On the other hand, the shorter and more fragmented is our sleep, the more difficult it is for us to maintain wakefulness and stable cognitive performance the next day. This relationship between wakefulness and subsequent sleep becomes especially apparent after sleep deprivation or during chronic sleep restriction, which is experienced by millions of people in our society, as well as in multiple neurological, respiratory and other chronic diseases. Invariably, poor sleep leads to fatigue, sleepiness, marked cognitive deficits and impaired mood. The crucial question is what happens to the brain after a period of being awake or asleep, and where in the brain and why do these changes occur. This review summarizes information about neurophysiological substrates of sleep homeostatic processes at the cellular and network levels. It is suggested that sensory, behavioral and cognitive deficits after sleep deprivation resulting from the imbalance between local and global neuronal interactions can be reversed only by physiological sleep.

Sonic hedgehog (Shh), a secreted glycoprotein factor, can activate the Shh pathway, which has been implicated in neuronal polarization involving neurite outgrowth. However, little evidence is available about the effect of Shh on neurite outgrowth in primary corticalneurons and its potential mechanism. Here, we revealed that Shh increased neurite outgrowth in primary corticalneurons, while the Shh pathway inhibitor (cyclopamine, CPM) partially suppressed Shh-induced neurite outgrowth. Similar results were found for the expressions of Shh and Patched genes in Shh-induced primary corticalneurons. Moreover, Shh increased the levels of brain-derived neurotrophic factor (BDNF) not only in lysates and in culture medium but also in the longest neurites of primary corticalneurons, which was partially blocked by CPM. In addition, blocking of BDNF action suppressed Shh-mediated neurite elongation in primary corticalneurons. In conclusion, these findings suggest that Shh promotes neurite outgrowth in primary corticalneurons at least partially through modulating BDNF expression.

Short-term memory refers to the ability to store small amounts of stimulus-specific information for a short period of time. It is supported by both fading and hidden memory processes. Fading memory relies on recurrent activity patterns in a neuronal network, whereas hidden memory is encoded using synaptic mechanisms, such as facilitation, which persist even when neurons fall silent. We have used a novel computational and optogenetic approach to investigate whether these same memory processes hypothesized to support pattern recognition and short-term memory in vivo, exist in vitro. Electrophysiological activity was recorded from primary cultures of dissociated rat corticalneurons plated on multielectrode arrays. Cultures were transfected with ChannelRhodopsin-2 and optically stimulated using random dot stimuli. The pattern of neuronal activity resulting from this stimulation was analyzed using classification algorithms that enabled the identification of stimulus-specific memories. Fading memories for different stimuli, encoded in ongoing neural activity, persisted and could be distinguished from each other for as long as 1 s after stimulation was terminated. Hidden memories were detected by altered responses of neurons to additional stimulation, and this effect persisted longer than 1 s. Interestingly, network bursts seem to eliminate hidden memories. These results are similar to those that have been reported from similar experiments in vivo and demonstrate that mechanisms of information processing and short-term memory can be studied using cultured neuronal networks, thereby setting the stage for therapeutic applications using this platform.

Full Text Available The study explored a modified primary culture system for fetal rat corticalneurons. Day E18 embryos from pregnant Sprague Dawley rats were microdissected under a stereoscope. To minimize enzymatic damage to the cultured neurons, we applied a sequential digestion protocol using papain and Dnase I. The resulting sifted cell suspension was seeded at a density of 50,000 cells per cm2 onto 0.1 mg/mL L-PLL-covered vessels. After a four-hour incubation in high-glucose Dulbecco’s Modified Eagle’s Medium (HG-DMEM to allow the neurons to adhere, the media was changed to neurobasal medium that was refreshed by changing half of the volume after three days followed by a complete medium change every week. The cells displayed progressively robust neurite extension, and nonneuronal-like cells could barely be detected by five days in vitro (DIV; cell growth was still substantial at 14 DIV. Neurons were identified by β-tubulin III immunofluorescence, and neuronal purity within the cultures was assessed at over 95% by both flow cytometry and by dark-field counting of β-tubulin III-positive cells. These results suggest that the protocol was successful and that the high purity of neurons in this system could be used as the basis for generating various cell models of neurological disease.

Full Text Available Local cortical circuits appear highly non-random, but the underlying connectivity rule remains elusive. Here, we analyze experimental data observed in layer 5 of rat neocortex and suggest a model for connectivity from which emerge essential observed non-random features of both wiring and weighting. These features include lognormal distributions of synaptic connection strength, anatomical clustering, and strong correlations between clustering and connection strength. Our model predicts that cortical microcircuits contain large groups of densely connected neurons which we call clusters. We show that such a cluster contains about one fifth of all excitatory neurons of a circuit which are very densely connected with stronger than average synapses. We demonstrate that such clustering plays an important role in the network dynamics, namely, it creates bistable neural spiking in small cortical circuits. Furthermore, introducing local clustering in large-scale networks leads to the emergence of various patterns of persistent local activity in an ongoing network activity. Thus, our results may bridge a gap between anatomical structure and persistent activity observed during working memory and other cognitive processes.

Full Text Available Subdural cortical stimulation (SuCS is an appealing method in the treatment of neurological disorders, and computational modeling studies of SuCS have been applied to determine the optimal design for electrotherapy. To achieve a better understanding of computational modeling on the stimulation effects of SuCS, the influence of anisotropic white matter conductivity on the activation of corticalneurons was investigated in a realistic head model. In this paper, we constructed pyramidal neuronal models (layers 3 and 5 that showed primary excitation of the corticospinal tract, and an anatomically realistic head model reflecting complex brain geometry. The anisotropic information was acquired from diffusion tensor magnetic resonance imaging (DT-MRI and then applied to the white matter at various ratios of anisotropic conductivity. First, we compared the isotropic and anisotropic models; compared to the isotropic model, the anisotropic model showed that neurons were activated in the deeper bank during cathodal stimulation and in the wider crown during anodal stimulation. Second, several popular anisotropic principles were adapted to investigate the effects of variations in anisotropic information. We observed that excitation thresholds varied with anisotropic principles, especially with anodal stimulation. Overall, incorporating anisotropic conductivity into the anatomically realistic head model is critical for accurate estimation of neuronal responses; however, caution should be used in the selection of anisotropic information.

Full Text Available Findings from recordings of human temporal cortical single neuron activity during several measures of language, including object naming and word reading are reviewed and related to changes in activity in the same neurons during recent verbal memory and verbal associative learning measures, in studies conducted during awake neurosurgery for the treatment of epilepsy. The proportion of neurons changing activity with language tasks was similar in either hemisphere. Dominant hemisphere activity was characterized by relative inhibition, some of which occurred during overt speech, possibly to block perception of one’s own voice. However, the majority seems to represent a dynamic network becoming active with verbal memory encoding and especially verbal learning, but inhibited during performance of overlearned language tasks. Individual neurons are involved in different networks for different aspects of language, including naming or reading and naming in different languages. The majority of the changes in activity were tonic sustained shifts in firing. Patterned phasic activity for specific language items was very infrequently recorded. Human single neuron recordings provide a unique perspective on the biologic substrate for language, for these findings are in contrast to many of the findings from other techniques for investigating this.

The postinhibitory rebound excitation is an intrinsic property of thalamic and corticalneurons that is implicated in a variety of normal and abnormal operations of neuronal networks, such as slow or fast brain rhythms during different states of vigilance as well as seizures. We used dual simultaneous intracellular recordings of thalamocortical neurons from the ventrolateral nucleus and neurons from the motor cortex, together with thalamic and cortical field potentials, to investigate the temporal relations between thalamic and cortical events during the rebound excitation that follows prolonged periods of stimulus-induced inhibition. Invariably, the rebound spike-bursts in thalamocortical cells occurred before the rebound depolarization in corticalneurons and preceded the peak of the depth-negative, rebound field potential in cortical areas. Also, the inhibitory-rebound sequences were more pronounced and prolonged in corticalneurons when elicited by thalamic stimuli, compared with cortical stimuli. The role of thalamocortical loops in the rebound excitation of corticalneurons was shown further by the absence of rebound activity in isolated cortical slabs. However, whereas thalamocortical neurons remained hyperpolarized after rebound excitation, because of the prolonged spike-bursts in inhibitory thalamic reticular neurons, the rebound depolarization in corticalneurons was prolonged, suggesting the role of intracortical excitatory circuits in this sustained activity. The role of intrathalamic events in triggering rebound cortical activity should be taken into consideration when analyzing information processes at the cortical level; at each step, corticothalamic volleys can set into action thalamic inhibitory neurons, leading to rebound spike-bursts that are transferred back to the cortex, thus modifying cortical activities. PMID:9811903

Full Text Available Despite our fine-grain anatomical knowledge of the cerebellar cortex, electrophysiological studies of circuit information processing over the last fifty years have been hampered by the difficulty of reliably assigning signals to identified cell types. We approached this problem by assessing the spontaneous activity signatures of identified cerebellar corticalneurones. A range of statistics describing firing frequency and irregularity were then used, individually and in combination, to build Gaussian Process Classifiers (GPC leading to a probabilistic classification of each neurone type and the computation of equi-probable decision boundaries between cell classes. Firing frequency statistics were useful for separating Purkinje cells from granular layer units, whilst firing irregularity measures proved most useful for distinguishing cells within granular layer cell classes. Considered as single statistics, we achieved classification accuracies of 72.5% and 92.7% for granular layer and molecular layer units respectively. Combining statistics to form twin-variate GPC models substantially improved classification accuracies with the combination of mean spike frequency and log-interval entropy offering classification accuracies of 92.7% and 99.2% for our molecular and granular layer models, respectively. A cross-species comparison was performed, using data drawn from anaesthetised mice and decerebrate cats, where our models offered 80% and 100% classification accuracy. We then used our models to assess non-identified data from awake monkeys and rabbits in order to highlight subsets of neurones with the greatest degree of similarity to identified cell classes. In this way, our GPC-based approach for tentatively identifying neurones from their spontaneous activity signatures, in the absence of an established ground-truth, nonetheless affords the experimenter a statistically robust means of grouping cells with properties matching known cell classes. Our

Substrate-bound gradients expressed in numerous spatio-temporal patterns play a crucial role during the development of complex neural circuits. A deeper understanding of the axon guidance mechanism is provided by studying the effect of a defined substrate-bound cue on a confined neural network. In this study, we constructed a discontinuous substrate-bound gradient to control neuronal cell position, the path of neurite growth, and axon directionality. A variety of gradient patterns, with slight changes in slope, width, and length were designed and fabricated by microcontact printing using laminin/poly-l-lysine (PLL) or PLL alone. The gradients were tested for neurite growth and their impact on axon guidance of embryonic rat corticalneurons. The neurite length was determined and the axon was evaluated by Tau-1 immunostaining. We found that the microgradients of laminin/PLL and PLL directed neurons' adhesion, differentially controlled the neurite growth, and guided up to 84% of the axons. The effect of the protein micropattern on axon guidance and neurite growth depended on the protein and geometric parameters used. Our approach proved to be very successful in guiding axons of single multipolar neurons with very high efficiency. It could thereby be useful to engineer defined neural networks for analyzing signal processing of functional circuits, as well as to unravel fundamental questions of the axon guidance mechanism.

Full Text Available The wide range of time scales involved in neural excitability and synaptic transmission might lead to ongoing change in the temporal structure of responses to recurring stimulus presentations on a trial-to-trial basis. This is probably the most severe biophysical constraint on putative time-based primitives of stimulus representation in neuronal networks. Here we show that in spontaneously developing large-scale random networks of corticalneurons in vitro the order in which neurons are recruited following each stimulus is a naturally emerging representation primitive that is invariant to significant temporal changes in spike times. With a relatively small number of randomly sampled neurons, the information about stimulus position is fully retrievable from the recruitment order. The effective connectivity that makes order-based representation invariant to time warping is characterized by the existence of stations through which activity is required to pass in order to propagate further into the network. This study uncovers a simple invariant in a noisy biological network in vitro; its applicability under in vivo constraints remains to be seen.

Premature infants are at risk for bilirubin-associated brain damage. In cell cultures bilirubin causes neuronal apoptosis and necrosis. Ibuprofen is used to close the ductus arteriosus, and is often given when hyperbilirubinemia is at its maximum. Ibuprofen is known to interfere with bilirubin-albumin binding. We hypothesized that bilirubin toxicity to cultured rat embryonic corticalneurons is augmented by coincubation with ibuprofen. Incubation with ibuprofen above a concentration of 125 microg/mL reduced cell viability, measured by methylthiazole tetrazolium reduction, to 68% of controls (p < 0.05). Lactate dehydrogenase (LDH) release increased from 29 to 38% (p < 0.01). The vehicle solution did not affect cell viability. Coincubation with 10 microM unconjugated bilirubin (UCB)/human serum albumin in a molar ratio of 3:1 and 250 microg/mL ibuprofen caused additional loss of cell viability and increased LDH release (p < 0.01), DNA fragmentation, and activated caspase-3. Preincubation with the pan-caspase inhibitor z-val-ala-asp-fluoromethyl ketone abolished ibuprofen- and UCB-induced DNA fragmentation. The study demonstrates that bilirubin in low concentration of 10 microM reduces neuron viability and ibuprofen increases this effect. Apoptosis is the underlying cell death mechanism.

Full Text Available We aimed to explore the cerebellar cortical inputs from two spinocerebellar pathways, the spinal border cell-component of the ventral spinocerebellar tract (SBC-VSCT and the dorsal spinocerebellar tract (DSCT, respectively, in the sublobule C1 of the cerebellar posterior lobe. The two pathways were activated by electrical stimulation of the contralateral lateral funiculus (coLF and the ipsilateral LF (iLF at lower thoracic levels. Most granule cells in sublobule C1 did not respond at all but part of the granule cell population displayed high-intensity responses to either coLF or iLF stimulation. As a rule, Golgi cells and Purkinje cell simple spikes responded to input from both LFs, although Golgi cells could be more selective. In addition, a small population of granule cells responded to input from both the coLF and the iLF. However, in these cases, similarities in the temporal topography and magnitude of the responses suggested that the same axons were stimulated from the two LFs, i.e. that the axons of individual spinocerebellar neurons could be present in both funiculi. This was also confirmed for a population of spinal neurons located within known locations of SBC-VSCT neurons and dorsal horn DSCT neurons. We conclude that bilateral spinocerebellar responses can occur in cerebellar granule cells, but the VSCT and DSCT systems that provide the input can also be organized bilaterally. The implications for the traditional functional separation of VSCT and DSCT systems and the issue whether granule cells primarily integrate functionally similar information or not are discussed.

Mutations of MECP2 cause Rett syndrome (RTT), a neurodevelopmental disorder leading to loss of motor and cognitive functions, impaired social interactions, and seizure at young ages. Defects of neuronal circuit development and function are thought to be responsible for the symptoms of RTT. The majority of RTT patients show recurrent seizures, indicating that neuronal hyperexcitation is a common feature of RTT. However, mechanisms underlying hyperexcitation in RTT are poorly understood. Here we show that deletion of Mecp2 from cortical excitatory neurons but not forebrain inhibitory neurons in the mouse leads to spontaneous seizures. Selective deletion of Mecp2 from excitatory but not inhibitory neurons in the forebrain reduces GABAergic transmission in layer 5 pyramidal neurons in the prefrontal and somatosensory cortices. Loss of MeCP2 from cortical excitatory neurons reduces the number of GABAergic synapses in the cortex, and enhances the excitability of layer 5 pyramidal neurons. Using single-cell deletion of Mecp2 in layer 2/3 pyramidal neurons, we show that GABAergic transmission is reduced in neurons without MeCP2, but is normal in neighboring neurons with MeCP2. Together, these results suggest that MeCP2 in cortical excitatory neurons plays a critical role in the regulation of GABAergic transmission and cortical excitability. PMID:24523563

The Working Memory model of human memory, first introduced by Baddeley and Hitch (1974), has been one of the most influential psychological constructs in cognitive psychology and human neuroscience. However the neuronal correlates of core components of this model have yet to be fully elucidated. Here we present data from two studies where human temporal cortical single neuron activity was recorded during tasks differentially affecting the maintenance component of verbal working memory. In Study One we vary the presence or absence of distracting items for the entire period of memory storage. In Study Two we vary the duration of storage so that distractors filled all, or only one-third of the time the memory was stored. Extracellular single neuron recordings were obtained from 36 subjects undergoing awake temporal lobe resections for epilepsy, 25 in Study one, 11 in Study two. Recordings were obtained from a total of 166 lateral temporal cortex neurons during performance of one of these two tasks, 86 study one, 80 study two. Significant changes in activity with distractor manipulation were present in 74 of these neurons (45%), 38 Study one, 36 Study two. In 48 (65%) of those there was increased activity during the period when distracting items were absent, 26 Study One, 22 Study Two. The magnitude of this increase was greater for Study One, 47.6%, than Study Two, 8.1%, paralleling the reduction in memory errors in the absence of distracters, for Study One of 70.3%, Study Two 26.3% These findings establish that human lateral temporal cortex is part of the neural system for working memory, with activity during maintenance of that memory that parallels performance, suggesting it represents active rehearsal. In 31 of these neurons (65%) this activity was an extension of that during working memory encoding that differed significantly from the neural processes recorded during overt and silent language tasks without a recent memory component, 17 Study one, 14 Study two

Full Text Available BACKGROUND: Sirtuins (Sirt, a family of nicotinamide adenine nucleotide (NAD dependent deacetylases, are implicated in energy metabolism and life span. Among the known Sirt isoforms (Sirt1-7, Sirt3 was identified as a stress responsive deacetylase recently shown to play a role in protecting cells under stress conditions. Here, we demonstrated the presence of Sirt3 in neurons, and characterized the role of Sirt3 in neuron survival under NMDA-induced excitotoxicity. METHODOLOGY/PRINCIPAL FINDINGS: To induce excitotoxic injury, we exposed primary cultured mouse corticalneurons to NMDA (30 µM. NMDA induced a rapid decrease of cytoplasmic NAD (but not mitochondrial NAD in neurons through poly (ADP-ribose polymerase-1 (PARP-1 activation. Mitochondrial Sirt3 was increased following PARP-1 mediated NAD depletion, which was reversed by either inhibition of PARP-1 or exogenous NAD. We found that massive reactive oxygen species (ROS produced under this NAD depleted condition mediated the increase in mitochondrial Sirt3. By transfecting primary neurons with a Sirt3 overexpressing plasmid or Sirt3 siRNA, we showed that Sirt3 is required for neuroprotection against excitotoxicity. CONCLUSIONS: This study demonstrated for the first time that mitochondrial Sirt3 acts as a prosurvival factor playing an essential role to protect neurons under excitotoxic injury.

Neuronal density, neuron size, and the number of neurons under 1 mm2 of cerebral cortical surface area were measured in the right pre-frontal cortex of Albert Einstein and five elderly control subjects. Measurement of neuronal density used the optical dissector technique on celloidin-embedded cresyl violet-stained sections. The neurons counted provided a systematic random sample for the measurement of cell body cross-sectional area. Einstein's cortex did not differ from the control subjects in the number of neurons under 1 mm2 of cerebral cortex or in mean neuronal size. Because Einstein's cortex was thinner than the controls he had a greater neuronal density.

In patients with higher brain dysfunction (HBD) after mild traumatic brain injury (MTBI), diagnostic imaging of corticalneuron loss in the frontal lobes was studied using SPECT with (123)I-iomazenil (IMZ), as a radioligand for central benzodiazepine receptor (BZR). Statistical imaging analysis using three-dimensional stereotactic surface projections (3D-SSP) for (123)I-IMZ SPECT was performed in 17 patients. In all patients with HBD defined by neuropsychological tests, corticalneuron loss was indicated in the bilateral medial frontal lobes in 14 patients (83 %). A comparison between the group of 17 patients and the normal database demonstrated common areas of corticalneuron loss in the bilateral medial frontal lobes involving the medial frontal gyrus (MFG) and the anterior cingulate gyrus (ACG). In an assessment of corticalneuron loss in the frontal medial cortex using the stereotactic extraction estimation (SEE) method (level 3), significant corticalneuron loss was observed within bilateral MFG in 9 patients and unilateral MFG in 4, and bilateral ACG in 12 and unilateral ACG in 3. Fourteen patients showed significant corticalneuron loss in bilateral MFG or ACG. In patients with MTBI, HBD seemed to correlate with selective corticalneuron loss within the bilateral MFG or ACG where the responsible lesion could be. 3D-SSP and SEE level 3 analysis for (123)I-IMZ SPECT could be valuable for diagnostic imaging of HBD after MTBI.

Matrix metalloproteinases and a disintegrin and metalloproteinases are members of the zinc endopeptidases, which cleave components of the extracellular matrix as well as cell surface proteins resulting in degradation or release of biologically active fragments. Surface ectodomain shedding affects numerous biological processes, including survival, axon outgrowth, axon guidance, and synaptogenesis. In this study, we evaluated the role of metalloproteinases in regulating cortical neurite growth. We found that treatment of mature corticalneurons with pan-metalloproteinase inhibitors or with tissue inhibitors of metalloproteinase-3 reduced neurite outgrowth. Through mass spectrometry, we characterized the metalloproteinase-sensitive cell surface proteome of mature corticalneurons. Members of the IgLON family of glycosylphosphatidylinositol-anchored neural cell adhesion molecules were identified and validated as proteins that were shed from the surface of mature corticalneurons in a metalloproteinase-dependent manner. Introduction of two members of the IgLON family, neurotrimin and NEGR1, in early embryonic neurons was sufficient to confer sensitivity to metalloproteinase inhibitors in neurite outgrowth assays. Outgrowth experiments on immobilized IgLON proteins revealed a role for all IgLON family members in promoting neurite extension from corticalneurons. Together, our findings support a role for metalloproteinase-dependent shedding of IgLON family members in regulating neurite outgrowth from mature corticalneurons.

Matrix metalloproteinases and a disintegrin and metalloproteinases are members of the zinc endopeptidases, which cleave components of the extracellular matrix as well as cell surface proteins resulting in degradation or release of biologically active fragments. Surface ectodomain shedding affects numerous biological processes, including survival, axon outgrowth, axon guidance, and synaptogenesis. In this study, we evaluated the role of metalloproteinases in regulating cortical neurite growth. We found that treatment of mature corticalneurons with pan-metalloproteinase inhibitors or with tissue inhibitors of metalloproteinase-3 reduced neurite outgrowth. Through mass spectrometry, we characterized the metalloproteinase-sensitive cell surface proteome of mature corticalneurons. Members of the IgLON family of glycosylphosphatidylinositol-anchored neural cell adhesion molecules were identified and validated as proteins that were shed from the surface of mature corticalneurons in a metalloproteinase-dependent manner. Introduction of two members of the IgLON family, neurotrimin and NEGR1, in early embryonic neurons was sufficient to confer sensitivity to metalloproteinase inhibitors in neurite outgrowth assays. Outgrowth experiments on immobilized IgLON proteins revealed a role for all IgLON family members in promoting neurite extension from corticalneurons. Together, our findings support a role for metalloproteinase-dependent shedding of IgLON family members in regulating neurite outgrowth from mature corticalneurons. PMID:25538237

Neuronal migration is integral to the development of the cerebral cortex and higher brain function. Corticalneuron migration defects lead to mental disorders such as lissencephaly and epilepsy. Interaction of neurons with their extracellular environment regulates corticalneuron migration through cell surface receptors. However, it is unclear how the signals from extracellular matrix proteins are transduced intracellularly. We report here that mouse embryos lacking the Ras family guanine nucleotide exchange factor, C3G (Rapgef1, Grf2), exhibit a corticalneuron migration defect resulting in a failure to split the preplate into marginal zone and subplate and a failure to form a cortical plate. C3G-deficient corticalneurons fail to migrate. Instead, they arrest in a multipolar state and accumulate below the preplate. The basement membrane is disrupted and radial glial processes are disorganised and lack attachment in C3G-deficient brains. C3G is activated in response to reelin in corticalneurons, which, in turn, leads to activation of the small GTPase Rap1. In C3G-deficient cells, Rap1 GTP loading in response to reelin stimulation is reduced. In conclusion, the Ras family regulator C3G is essential for two aspects of cortex development, namely radial glial attachment and neuronal migration.

Full Text Available Injury to the human central nervous system can lead to devastating consequences due to its poor ability to self-repair. Neural transplantation aimed at replacing lost neurons and restore functional circuitry has proven to be a promising therapeutical avenue. We previously reported in adult rodent animal models with cortical lesions that grafted fetal corticalneurons could effectively re-establish specific patterns of projections and synapses. The current study was designed to provide a detailed characterization of the spatio-temporal in vivo development of fetal cortical transplanted cells within the lesioned adult motor cortex and their corresponding axonal projections. We show here that as early as two weeks after grafting, cortical neuroblasts transplanted into damaged adult motor cortex developed appropriate projections to cortical and subcortical targets. Grafted cells initially exhibited characteristics of immature neurons, which then differentiated into mature neurons with appropriate cortical phenotypes where most were glutamatergic and few were GABAergic. All cortical subtypes identified with the specific markers CTIP2, Cux1, FOXP2 and Tbr1 were generated after grafting as evidenced with BrdU co-labeling.The set of data provided here is of interest as it sets biological standards for future studies aimed at replacing fetal cells with embryonic stem cells as a source of corticalneurons.

Heterozygous mutations or deletions in the human Euchromatin histone methyltransferase 1 (EHMT1) gene cause Kleefstra syndrome, a neurodevelopmental disorder that is characterized by autistic-like features and severe intellectual disability (ID). Neurodevelopmental disorders including ID and autism may be related to deficits in activity-dependent wiring of brain circuits during development. Although Kleefstra syndrome has been associated with dendritic and synaptic defects in mice and Drosophila, little is known about the role of EHMT1 in the development of corticalneuronal networks. Here we used micro-electrode arrays and whole-cell patch-clamp recordings to investigate the impact of EHMT1 deficiency at the network and single cell level. We show that EHMT1 deficiency impaired neural network activity during the transition from uncorrelated background action potential firing to synchronized network bursting. Spontaneous bursting and excitatory synaptic currents were transiently reduced, whereas miniature excitatory postsynaptic currents were not affected. Finally, we show that loss of function of EHMT1 ultimately resulted in less regular network bursting patterns later in development. These data suggest that the developmental impairments observed in EHMT1-deficient networks may result in a temporal misalignment between activity-dependent developmental processes thereby contributing to the pathophysiology of Kleefstra syndrome. PMID:27767173

Full Text Available Presenilins are the major causative genes of familial Alzheimer's disease (AD. Our previous study has demonstrated essential roles of presenilins in memory and neuronal survival. Here, we explore further how loss of presenilins results in age-related, progressive neurodegeneration in the adult cerebral cortex, where the pathogenesis of AD occurs. To circumvent the requirement of presenilins for embryonic development, we used presenilin conditional double knockout (Psen cDKO mice, in which presenilin inactivation is restricted temporally and spatially to excitatory neurons of the postnatal forebrain beginning at 4 weeks of age. Increases in the number of degenerating (Fluoro-Jade B+, 7.6-fold and apoptotic (TUNEL+, 7.4-fold neurons, which represent approximately 0.1% of all corticalneurons, were first detected at 2 months of age when there is still no significant loss of corticalneurons and volume in Psen cDKO mice. By 4 months of age, significant loss of corticalneurons (approximately 9% and gliosis was found in Psen cDKO mice. The apoptotic cell death is associated with caspase activation, as shown by increased numbers of cells immunoreactive for active caspases 9 and 3 in the Psen cDKO cortex. The vulnerability of corticalneurons to loss of presenilins is region-specific with corticalneurons in the lateral cortex most susceptible. Compared to the neocortex, the increase in apoptotic cell death and the extent of neurodegeneration are less dramatic in the Psen cDKO hippocampus, possibly in part due to increased neurogenesis in the aging dentate gyrus. Neurodegeneration is also accompanied with mitochondrial defects, as indicated by reduced mitochondrial density and altered mitochondrial size distribution in aging Psen corticalneurons. Together, our findings show that loss of presenilins in corticalneurons causes apoptotic cell death occurring in a very small percentage of neurons, which accumulates over time and leads to substantial loss

How are neurons distributed along the cortical surface and across functional areas? Here we use the isotropic fractionator (Herculano-Houzel and Lent, 2005) to analyze the distribution of neurons across the entire isocortex of the mouse, divided into 18 functional areas defined anatomically. We find that the number of neurons underneath a surface area (the N/A ratio) varies 4.5-fold across functional areas and neuronal density varies 3.2-fold. The face area of S1 contains the most neurons, followed by motor cortex and the primary visual cortex. Remarkably, while the distribution of neurons across functional areas does not accompany the distribution of surface area, it mirrors closely the distribution of cortical volumes-with the exception of the visual areas, which hold more neurons than expected for their volume. Across the non-visual cortex, the volume of individual functional areas is a shared linear function of their number of neurons, while in the visual areas, neuronal densities are much higher than in all other areas. In contrast, the 18 functional areas cluster into three different zones according to the relationship between the N/A ratio and cortical thickness and neuronal density: these three clusters can be called visual, sensory, and, possibly, associative. These findings are remarkably similar to those in the human cerebral cortex (Ribeiro et al., 2013) and suggest that, like the human cerebral cortex, the mouse cerebral cortex comprises two zones that differ in how neurons form the cortical volume, and three zones that differ in how neurons are distributed underneath the cortical surface, possibly in relation to local differences in connectivity through the white matter. Our results suggest that beyond the developmental divide into visual and non-visual cortex, functional areas initially share a common distribution of neurons along the parenchyma that become delimited into functional areas according to the pattern of connectivity established later.

The slow cortical oscillation is the major brain rhythm occurring during sleep, and has been the object of thorough investigation for over thirty years. Despite all these efforts, the function and the neuronal mechanisms behind slow cortical rhythms remain only partially understood. In this review

The slow cortical oscillation is the major brain rhythm occurring during sleep, and has been the object of thorough investigation for over thirty years. Despite all these efforts, the function and the neuronal mechanisms behind slow cortical rhythms remain only partially understood. In this review w

Acidosis impairs cognitions and behaviors presumably by acidification-induced changes in neuronal metabolism. Cortical GABAergic neurons are vulnerable to pathological factors and their injury leads to brain dysfunction. How acidosis induces GABAergic neuron injury remains elusive. As the glia cells and neurons interact each other, we intend to examine the role of the astrocytes in acidosis-induced GABAergic neuron injury. Experiments were done at GABAergic cells and astrocytes in mouse cortical slices. To identify astrocytic involvement in acidosis-induced impairment, we induced the acidification in single GABAergic neuron by infusing proton intracellularly or in both neurons and astrocytes by using proton extracellularly. Compared the effects of intracellular acidification and extracellular acidification on GABAergic neurons, we found that their active intrinsic properties and synaptic outputs appeared more severely impaired in extracellular acidosis than intracellular acidosis. Meanwhile, extracellular acidosis deteriorated glutamate transporter currents on the astrocytes and upregulated excitatory synaptic transmission on the GABAergic neurons. Moreover, the antagonists of glutamate NMDA-/AMPA-receptors partially reverse extracellular acidosis-induced injury in the GABAergic neurons. Our studies suggest that acidosis leads to the dysfunction of cortical GABAergic neurons by astrocyte-mediated excitotoxicity, in addition to their metabolic changes as indicated previously.

The migration of projection neurons from its birthplace in the subventricular zone to their final destination in the cortical plate is a complex process that requires a series of highly coordinated cellular events. Amongst the key factors involved in the processes are modulators of cytoskeletal dynamics, as well as cellular membrane traffic. Members of the small GTPases family responsible for the latter process, the Rabs and Arfs, have been recently implicated in corticalneuron migration. Rab5 and Rab11, which are key modulators of endocytosis and endocytic recycling respectively, ensure proper surface expression and distribution of N-cadherin, a key adhesion protein that tethers migrating neurons to the radial glia fiber tracts during pia-directed migration. Rab7, which is associated with lysosomal biogenesis and function, is important for the final step of terminal translocation when N-cadherin is downregulated by lysosomal degradation. Arf6 activity, which is known to be important in neuronal processes outgrowth, may negatively impact the multipolar-bipolar transition of corticalneurons undergoing radial migration, but the downstream effector of Arf6 in this regard is not yet known. In addition to the above, members of the Arl family which have been recently shown to be important in radial glia scaffold formation, would also be important for corticalneuron migration. In this short review, we discuss recent advances in our understanding of the importance of membrane traffic regulated by the Rab, Arf, and Arl family members in corticalneuron migration.

This video will guide you through the process of culturing rat corticalneurons in the presence of a glial feeder layer, a system known as a bilaminar or co-culture model. This system is suitable for a variety of experimental needs requiring either a glass or plastic growth substrate and can also be used for culture of other types of neurons. Rat corticalneurons obtained from the late embryonic stage (E17) are plated on glass coverslips or tissue culture dishes facing a feeder layer of glia grown on dishes or plastic coverslips (known as Thermanox), respectively. The choice between the two configurations depends on the specific experimental technique used, which may require, or not, that neurons are grown on glass (e.g. calcium imaging versus Western blot). The glial feeder layer, an astroglia-enriched secondary culture of mixed glia, is separately prepared from the cortices of newborn rat pups (P2-4) prior to the neuronal dissection. A major advantage of this culture system as compared to a culture of neurons only is the support of neuronal growth, survival, and differentiation provided by trophic factors secreted from the glial feeder layer, which more accurately resembles the brain environment in vivo. Furthermore, the co-culture can be used to study neuronal-glial interactions(1). At the same time, glia contamination in the neuronal layer is prevented by different means (low density culture, addition of mitotic inhibitors, lack of serum and use of optimized culture medium) leading to a virtually pure neuronal layer, comparable to other established methods(1-3). Neurons can be easily separated from the glial layer at any time during culture and used for different experimental applications ranging from electrophysiology(4), cellular and molecular biology(5-8), biochemistry(5), imaging and microscopy(4,6,7,9,10). The primary neurons extend axons and dendrites to form functional synapses(11), a process which is not observed in neuronal cell lines, although some

Cortical layer 5 (L5) pyramidal neurons integrate inputs from many sources and distribute outputs to cortical and subcortical structures. Previous studies demonstrate two L5 pyramid types: cortico-cortical (CC) and cortico-subcortical (CS). We characterize connectivity and function of these cell types in mouse primary visual cortex and reveal a new subtype. Unlike previously described L5 CC and CS neurons, this new subtype does not project to striatum [cortico-cortical, non-striatal (CC-NS)] and has distinct morphology, physiology, and visual responses. Monosynaptic rabies tracing reveals that CC neurons preferentially receive input from higher visual areas, while CS neurons receive more input from structures implicated in top-down modulation of brain states. CS neurons are also more direction-selective and prefer faster stimuli than CC neurons. These differences suggest distinct roles as specialized output channels, with CS neurons integrating information and generating responses more relevant to movement control and CC neurons being more important in visual perception.

DNA damage, as an important initiator of neuronal cell death, has been implicated in numerous neurodegenerative conditions. We previously delineated several pathways that control embryonic corticalneuronal cell death evoked by the DNA-damaging agent, camptothecin. The topisomerase-1 inhibitor, camptothecin, has been shown to induce corticalneuronal cell death in a reproducible and synchronistic manner. Primary embryonic neuronal cell culture corticalneurons were prepared. In the study, the survival % of neurons in camptothecin P38 group, after 6 hours (85%), 24 hours (64%) and 48 hours (50%), compared to camptothecin ATF-2 and P38 group after 4 hours (97 and 95%), have been significantly lower, and the expression % of neurons in camptothecin P38 group , after 6 hours (20%), 24 hours (40%) and 48 hours (55%), compared to camptothecin ATF-2 and P38 group after 4 hours (5 and 3%) have been significant lower (pATF-2 group after 24hours (30%), have been significant lower (pATF-2 in embryonic corticalneurons following DNA damage.

Networks of corticalneurons were grown over multi electrode arrays to enable simultaneous measurement of signals from multiple neurons. We described functional connectivity in these networks by relationships be¬tween individual electrodes, based on conditional firing probabilities. In this study we

Full Text Available A key question in understanding AD is whether extracellular Abeta deposition of parenchymal amyloid plaques or intraneuronal Abeta accumulation initiates the AD process. Amyloid precursor protein (APP is endocytosed from the cell surface into endosomes where it is cleaved to produce soluble Abeta which is then released into the brain interstitial fluid. Intraneuronal Abeta accumulation is hypothesized to predominate from the neuronal uptake of this soluble extracellular Abeta rather than from ER/Golgi processing of APP. We demonstrate that substitution of the two adjacent histidine residues of Abeta40 results in a significant decrease in its binding with PC12 cells and mouse cortical/hippocampal neurons. These substitutions also result in a dramatic enhancement of both thioflavin-T positive fibril formation and binding to preformed Abeta fibrils while maintaining its plaque-binding ability in AD transgenic mice. Hence, alteration of the histidine domain of Abeta prevented neuronal binding and drove Abeta to enhanced fibril formation and subsequent amyloid plaque deposition--a potential mechanism for removing toxic species of Abeta. Substitution or even masking of these Abeta histidine residues might provide a new therapeutic direction for minimizing neuronal uptake and subsequent neuronal degeneration and maximizing targeting to amyloid plaques.

It is accepted that the mechanisms of low level laser therapy (LLLT) involves photons that are absorbed in the mitochondria of cells and lead to increase of mitochondrial metabolism resulting in more electron transport, increase of mitochondrial membrane potential, and more ATP production. Intracellular calcium changes are seen that correlate with mitochondrial stimulation. The situation with two other intermediates is more complex however: reactive oxygen species (ROS) and nitric oxide (NO). Evidence exists that low levels of ROS are produced by LLLT in normal cells that can be beneficial by (for instance) activating NF-kB. However high fluences of light can produce large amounts of ROS that can damage the cells. In oxidatively stressed cells the situation may be different. We exposed primary cultured corticalneurons to hydrogen peroxide (H2O2) or cobalt chloride (CoCl2) oxidative insults in the presence or absence of LLLT (810-nm laser at 0.3 or 3 J/cm2). Cell viability of corticalneurons was determined by lactate dehydrogenase assay. ROS in neurons was detected using an ROS probe, MitoRox with confocal microscopy. Results showed that LLLT dose-dependently reversed ROS production and protected corticalneurons against H2O2 or CoCl2 induced oxidative injury in cultured corticalneurons. Conclusion: LLLT can protect corticalneurons against oxidative stress by reversing the levels of ROS.

Neuronal migration is a fundamental process during the development of the cerebral cortex and is regulated by cytoskeletal components. Microtubule dynamics can be modulated by posttranslational modifications to tubulin subunits. Acetylation of α-tubulin at lysine 40 is important in regulating microtubule properties, and this process is controlled by acetyltransferase and deacetylase. MEC-17 is a newly discovered α-tubulin acetyltransferase that has been found to play a major role in the acetylation of α-tubulin in different species in vivo. However, the physiological function of MEC-17 during neural development is largely unknown. Here, we report that MEC-17 is critical for the migration of corticalneurons in the rat. MEC-17 was strongly expressed in the cerebral cortex during development. MEC-17 deficiency caused migratory defects in the cortical projection neurons and interneurons, and perturbed the transition of projection neurons from the multipolar stage to the unipolar/bipolar stage in the intermediate zone of the cortex. Furthermore, knockdown of α-tubulin deacetylase HDAC6 or overexpression of tubulin(K40Q) to mimic acetylated α-tubulin could reduce the migratory and morphological defects caused by MEC-17 deficiency in cortical projection neurons. Thus, MEC-17, which regulates the acetylation of α-tubulin, appears to control the migration and morphological transition of corticalneurons. This finding reveals the importance of MEC-17 and α-tubulin acetylation in cortical development.

The formation of axon-dendrite polarity is crucial for neuron to make the proper information flow within the brain. Although the processes of neuronal polarity formation have been extensively studied using neurons in dissociated culture, the corresponding developmental processes in vivo are still unclear. Here, we illuminate the initial steps of morphological polarization of excitatory corticalneurons in situ, by sparsely labeling their neuroepithelial progenitors using in utero electroporation and then examining their neuronal progeny in brain sections and in slice cultures. Morphological analysis showed that an axon-like long tangential process formed in progeny cells in the intermediate zone (IZ). Time-lapse imaging analysis using slice culture revealed that progeny cells with multipolar shape, after alternately extending and retracting their short processes for several hours, suddenly elongated a long process tangentially. These cells then transformed into a bipolar shape, extending a pia-directed leading process, and migrated radially leaving the tangential process behind, which gave rise to an "L-shaped" axon. Our findings suggest that neuronal polarity in these cells is established de novo from a nonpolarized stage in vivo and indicate that excitatory corticalneurons with multipolar shape in the IZ initiate axon outgrowth before radial migration into the cortical plate.

Translating neuroprotective treatments from discovery in cell and animal models to the clinic has proven challenging. To reduce the gap between basic studies of neurotoxicity and neuroprotection and clinically relevant therapies, we developed a human corticalneuron culture system from human embryonic stem cells or human inducible pluripotent stem cells that generated both excitatory and inhibitory neuronal networks resembling the composition of the human cortex. This methodology used timed administration of retinoic acid to FOXG1(+) neural precursor cells leading to differentiation of neuronal populations representative of the six cortical layers with both excitatory and inhibitory neuronal networks that were functional and homeostatically stable. In human corticalneuronal cultures, excitotoxicity or ischemia due to oxygen and glucose deprivation led to cell death that was dependent on N-methyl-D-aspartate (NMDA) receptors, nitric oxide (NO), and poly(ADP-ribose) polymerase (PARP) (a cell death pathway called parthanatos that is distinct from apoptosis, necroptosis, and other forms of cell death). Neuronal cell death was attenuated by PARP inhibitors that are currently in clinical trials for cancer treatment. This culture system provides a new platform for the study of human cortical neurotoxicity and suggests that PARP inhibitors may be useful for ameliorating excitotoxic and ischemic cell death in human neurons.

Full Text Available The vast majority of cortical GABAergic neurons can be defined by parvalbumin, somatostatin or calretinin expression. In most mammalians parvalbumin and somatostatin interneurons have constant proportions, each representing 5-7% of the total neuron number. In contrast, there is a 3 fold increase in the proportion of calretinin interneurons, which do not exceed 4% in rodents and reach 12% in higher order areas of primate cerebral cortex. In rodents almost all parvalbumin and somatostatin interneurons originate from the medial part of the subpallial proliferative structure, the ganglionic eminence (GE, while almost all calretinin interneurons originate from its caudal part. The spatial pattern of cortical GABAergic neurons origin from the GE is preserved in the monkey and human brain. However, it could be expected that the evolution is changing developmental rules to enable considerable expansion of calretinin interneuron population. During the early fetal period in primates cortical GABAergic neurons are almost entirely generated in the subpallium, as in rodents. Already at that time the primate caudal ganglionic eminence (CGE shows a relative increase in size and production of calretinin interneurons. During the second trimester of gestation, that is the main neurogenetic stage in primates without clear correlates found in rodents, the pallial production of cortical GABAergic neurons together with the extended persistence of the GE is observed. We propose that the CGE could be the main source of calretinin interneurons for the posterior and lateral cortical regions, but not for the frontal cortex. The associative granular frontal cortex represents around one third of the cortical surface and contains almost half of cortical calretinin interneurons. The majority of calretinin interneurons destined for the frontal cortex could be generated in the pallium, especially in the newly evolved outer subventricular zone that becomes the main pool of

A subpopulation of chick retinal projection neurons becomes tetraploid during development, an event prevented by blocking antibodies against p75 neurotrophin receptor (p75(NTR)). We have used an optimized flow cytometric assay, based on the analysis of unfixed brain cell nuclei, to study whether p75(NTR)-dependent neuronal tetraploidization takes place in the cerebral cortex, giving rise to projection neurons as well. We show that 3% of neurons in both murine neocortex and chick telencephalic derivatives are tetraploid, and that in the mouse ~85% of these neurons express the immediate early genes Erg-1 and c-Fos, indicating that they are functionally active. Tetraploid corticalneurons (65-80%) express CTIP2, a transcription factor specific for subcortical projection neurons in the mouse neocortex. During the period in which these neurons are born, p75(NTR) is detected in differentiating neurons undergoing DNA replication. Accordingly, p75(NTR)-deficient mice contain a reduced proportion of both NeuN and CTIP2-positive neocortical tetraploid neurons, thus providing genetic evidence for the participation of p75(NTR) in the induction of neuronal tetraploidy in the mouse neocortex. In the striatum tetraploidy is mainly associated with long-range projection neurons as well since ~80% of tetraploid neurons in this structure express calbindin, a marker of neostriatal-matrix spiny neurons, known to establish long-range projections to the substantia nigra and globus pallidus. In contrast, only 20% of tetraploid corticalneurons express calbindin, which is mainly expressed in layers II-III, where CTIP2 is absent. We conclude that tetraploidy mainly affects long-range projection neurons, being facilitated by p75(NTR) in the neocortex.

Full Text Available Introduction Cortical amygdaloid nucleus belongs to the corticomedial part of the amygdaloid complex. In this nucleus there are neurons that produce neuropetide Y. This peptide has important roles in sleeping, learning, memory, gastrointestinal regulation, anxiety, epilepsy, alcoholism and depression. Material and methods We investigated morphometric characteristics (numbers of primary dendrites, longer and shorter diameters of cell bodies and maximal radius of dendritic arborization of NPY immunoreactive neurons of human cortical amygdaloid nucleus on 6 male adult human brains, aged 46 to 77 years, by immunohistochemical avidin-biotin technique. Results Our investigation has shown that in this nucleus there is a moderate number of NPY immunoreactive neurons. 67% of found neurons were nonpyramidal, while 33% were pyramidal. Among the nonpyramidal neurons the dominant groups were multipolar neurons (41% - of which 25% were multipolar irregular, and 16% multipolar oval. Among the pyramidal neurons the dominant groups were the neurons with triangular shape of cell body (21%. All found NPY immunoreactive neurons (pyramidal and nonpyramidal altogether had intervals of values of numbers of primary dendrites 2 to 6, longer diameters of cell bodies 13 to 38 µm, shorter diameters of cell bodies 9 to 20 µm and maximal radius of dendritic arborization 50 to 340 µm. More than a half of investigated neurons (57% had 3 primary dendrites. Discussion and conclusion The other researchers did not find such percentage of pyramidal immunoreactive neurons in this amygdaloid nucleus. If we compare our results with the results of the ather researchers we can conclude that all pyramidal NPY immunoreactive neurons found in this human amygdaloid nucleus belong to the class I of neurons, and that all nonpyramidal NPY immunoreactive neurons belong to the class II of neurons described by other researchers. We suppose that all found pyramidal neurons were projectional.

Full Text Available Up to date, little is known about the repair mode of microdamage in osteonal cortical bone resulting from bone screw implantation. In this study, self-tapping titanium cortical bone screws were inserted into the tibial diaphyses of 24 adult male rabbits. The animals were sacrificed at 1 day, 2 weeks, 1 month and 2 months after surgery. Histomorphometric measurement and confocal microscopy were performed on basic fuchsin stained bone sections to examine the morphological characteristics of microdamage, bone resorption activity and spatial relationship between microdamage and bone resorption. Diffuse and linear cracks were coexisted in peri-screw bone. Intracortical bone resorption was significantly increased 2 weeks after screw installation and reach to the maximum at 1 month. There was no significant difference in bone resorption between 1-month and 2-months groups. Microdamage was significantly decreased within 1 month after surgery. Bone resorption was predisposed to occur in the region of <100 µm from the bone-screw interface, where had extensive diffuse damage mixed with linear cracks. Different patterns of resorption cavities appeared in peri-screw bone. These data suggest that 1 the complex microdamage composed of diffuse damage and linear cracks is a strong stimulator for initiating targeted bone remodeling; 2 bone resorption activities taking place on the surfaces of differently oriented Haversian and Volkmann canals work in a team for the repair of extensive microdamage; 3 targeted bone remodeling is a short-term reaction to microdamage and thereby it may not be able to remove all microdamage resulting from bone screw insertion.

Full Text Available Polymorphisms that alter serotonin transporter SERT expression and functionality increase the risks for autism and psychiatric traits. Here, we investigate how SERT controls serotonin signaling in developing CNS in mice. SERT is transiently expressed in specific sets of glutamatergic neurons and uptakes extrasynaptic serotonin during perinatal CNS development. We show that SERT expression in glutamatergic thalamocortical axons (TCAs dictates sensory map architecture. Knockout of SERT in TCAs causes lasting alterations in TCA patterning, spatial organizations of corticalneurons, and dendritic arborization in sensory cortex. Pharmacological reduction of serotonin synthesis during the first postnatal week rescues sensory maps in SERTGluΔ mice. Furthermore, knockdown of SERT expression in serotonin-producing neurons does not impair barrel maps. We propose that spatiotemporal SERT expression in non-serotonin-producing neurons represents a determinant in early life genetic programming of cortical circuits. Perturbing this SERT function could be involved in the origin of sensory and cognitive deficits associated with neurodevelopmental disorders.

The insecticidal and neurotoxic effects of pyrethroids result from prolonged sodium channel inactivation, which causes alterations in neuronal firing and communication. Previously, we determined the relative potencies of 11 type I and type II pyrethroid insecticides using microel...

L-valine is a branched-chain amino acid (BCAA) largely used as dietary integrator by athletes and involved in some inherited rare diseases such as maple syrup urine disease. This pathology is caused by an altered BCAA metabolism with the accumulation of toxic keto acids in tissues and body fluids with consequent severe neurological symptoms. In animal models of BCAA accumulation, increased oxidative stress levels and lipid peroxidation have been reported. The aim of this study was to analyze both whether high BCAA concentrations in neurons induce reactive oxygen species (ROS) production and whether, by performing electrophysiological recordings, the neuronal functional properties are modified. Our results demonstrate that in primary cortical cultures, a high dose of valine increases ROS production and provokes neuronal hyperexcitability because the action potential frequencies and the persistent sodium current amplitudes increase significantly compared to non-treated neurons. Since Baicalein, a flavone obtained from the Scutellaria root, has been shown to act as a strong antioxidant with neuroprotective effects, we evaluated its possible antioxidant activity in primary corticalneurons chronically exposed to L-valine. The preincubation of corticalneurons with Baicalein prevents the ROS production and is able to revert both the neuronal hyperexcitability and the increase of the persistent sodium current, indicating a direct correlation between the ROS production and the altered physiological parameters. In conclusion, our data show that the electrophysiological alterations of corticalneurons elicited by high valine concentration are due to the increase in ROS production, suggesting much caution in the intake of BCAA dietary integrators.

Full Text Available Unlike stereotypical neurotropic viruses, influenza A viruses have been detected in the brain tissues of human and animal models. To investigate the interaction between neurons and influenza A viruses, mouse corticalneurons were isolated, infected with human H1N1 influenza virus, and then examined for the production of various inflammatory molecules involved in immune response. We found that replication of the influenza virus in neurons was limited, although early viral transcription was not affected. Virus-induced neuron viability decreased at 6 h postinfection (p.i. but increased at 24 h p.i. depending upon the viral strain. Virus-induced apoptosis and cytopathy in primary corticalneurons were not apparent at 24 h p.i. The mRNA levels of inflammatory cytokines, chemokines, and type I interferons were upregulated at 6 h and 24 h p.i. These results indicate that the influenza virus induces inflammatory response in mouse primary corticalneurons with limited viral replication. The cytokines released in viral infection-induced neuroinflammation might play critical roles in influenza encephalopathy, rather than in viral replication-induced cytopathy.

We report a procedure for recording the simultaneous activity of single neurons distributed across five cortical areas in behaving monkeys. The procedure consists of a commercially available microdrive adapted to a commercially available neural data collection system. The critical advantage of this procedure is that, in each cortical area, a configuration of seven microelectrodes spaced 250–500 μm can be inserted transdurally and each can be moved independently in the z axis. For each microelectrode, the data collection system can record the activity of up to five neurons together with the local field potential (LFP). With this procedure, we normally monitor the simultaneous activity of 70–100 neurons while trained monkeys discriminate the difference in frequency between two vibrotactile stimuli. Approximately 20–60 of these neurons have response properties previously reported in this task. The neuronal recordings show good signal-to-noise ratio, are remarkably stable along a 1-day session, and allow testing several protocols. Microelectrodes are removed from the brain after a 1-day recording session, but are reinserted again the next day by using the same or different x-y microelectrode array configurations. The fact that microelectrodes can be moved in the z axis during the recording session and that the x-y configuration can be changed from day to day maximizes the probability of studying simultaneous interactions, both local and across distant cortical areas, between neurons associated with the different components of this task. PMID:18946031

Neuropeptide Y has been shown to inhibit the immunological activity of reactive microglia in the rat cerebral cortex, to reduce N-methyl-D-aspartate current (INMDA) in corticalneurons, and protect neurons. In this study, after primary cultured microglia from the cerebral cortex of rats were treated with lipopolysaccharide, interleukin-1β and tumor necrosis factor-α levels in the cell culture medium increased, and mRNA expression of these cytokines also increased. After primary cultured corticalneurons were incubated with the lipopolysaccharide-treated microg-lial conditioned medium, peak INMDA in neurons increased. These effects of lipopolysaccharide were suppressed by neuropeptide Y. After addition of the neuropeptide Y Y1 receptor antago-nist BIBP3226, the effects of neuropeptide Y completely disappeared. These results suggest that neuropeptide Y prevents excessive production of interleukin-1β and tumor necrosis factor-α by inhibiting microglial reactivity. This reduces INMDA in rat corticalneurons, preventing excitotoxic-ity, thereby protecting neurons.

The correlation between neuronal activity and cortical hemodynamics, namely, neurovascular coupling (NVC), is important to shed light on the mechanism of a variety of brain functions or neuronal diseases. NVC can be studied by simultaneously measuring neuronal activity and cortical hemodynamics. Consequently, noninvasive measurements of the NVC have been widely studied using both electroencephalography (EEG) and functional magnetic resonance imaging (fMRI). However, electromagnetic interference between EEG and fMRI is still a major problem. On the other hand, near-infrared spectroscopy (NIRS) is another promising tool for detecting cortical hemodynamics because it can be combined with EEG or magnetoencephalography (MEG) without any electromagnetic interference. Accordingly, in the present study, a simultaneous measurement system-combining an unshielded MEG using a two-dimensional gradiometer based on a low-T superconducting quantum interference device (SQUID) and an NIRS using nonmagnetic thin probes-was developed. This combined system was used to simultaneously measure both an auditory-evoked magnetic field and blood flow change in the auditory cortex. It was experimentally demonstrated that the combined unshielded MEG/NIRS system can simultaneously measure neuronal activity and cortical hemodynamics.

In this paper we address the issue of spontaneous bursting activity in corticalneuronal cultures and explain what might cause this collective behavior using computer simulations of two different neural network models. While the common approach to acivate a passive network is done by introducing

Cortical projection neurons exhibit diverse morphological, physiological, and molecular phenotypes, but it is unknown how many distinct types exist. Many projection cell phenotypes are associated with laminar fate (radial position), but each layer may also contain multiple types of projection cells.

Increased brain size is common in children with autism spectrum disorders. Here we propose that an increased number of cortical excitatory neurons may underlie the increased brain volume, minicolumn pathology and excessive network excitability, leading to sensory hyper-reactivity and seizures, which are often found in autism. We suggest that…

Full Text Available Nucleoside analogue reverse transcriptase inhibitor (NRTI, an integral component of highly active antiretroviral therapy (HAART, was widely used to inhibit HIV replication. Long-term exposure to NRTIs can result in mitochondrial toxicity which manifests as lipoatrophy, lactic acidosis, cardiomyopathy and myopathy, as well as polyneuropathy. But the cerebral neurotoxicity of NRTIs is still not well known partly due to the restriction of blood-brain barrier (BBB and the complex microenvironment of the central nervous system (CNS. In this study, the Balb/c mice were administered 50 mg/kg stavudine (D4T, 100 mg/kg zidovudine (AZT, 50 mg/kg lamivudine (3TC or 50 mg/kg didanosine (DDI per day by intraperitoneal injection, five days per week for one or four months, and primary corticalneurons were cultured and exposed to 25 µM D4T, 50 µM AZT, 25 µM 3TC or 25 µM DDI for seven days. Then, single neuron was captured from mouse cerebral cortical tissues by laser capture microdissection. Mitochondrial DNA (mtDNA levels of the primary cultured corticalneurons, and captured neurons or glial cells, and the tissues of brains and livers and muscles were analyzed by relative quantitative real-time PCR. The data showed that mtDNA did not lose in both NRTIs exposed cultured neurons and one month NRTIs treated mouse brains. In four months NRTIs treated mice, brain mtDNA levels remained unchanged even if the mtDNA levels of liver (except for 3TC and muscle significantly decreased. However, mtDNA deletion was significantly higher in the captured neurons from mtDNA unchanged brains. These results suggest that long-term exposure to NRTIs can result in mtDNA deletion in mouse corticalneurons.

Dietary supplementation has been studied as an approach to ameliorating deficits associated with aging and neurodegeneration. We undertook this pilot study to investigate the effects of coconut oil supplementation directly on corticalneurons treated with amyloid-β (Aβ) peptide in vitro. Our results indicate that neuron survival in cultures co-treated with coconut oil and Aβ is rescued compared to cultures exposed only to Aβ. Coconut oil co-treatment also attenuates Aβ-induced mitochondrial alterations. The results of this pilot study provide a basis for further investigation of the effects of coconut oil, or its constituents, on neuronal survival focusing on mechanisms that may be involved.

Fast synaptic inhibition sculpts all forms of cortical activity by means of a specialized connectivity pattern between highly heterogeneous inhibitory interneurons and principal excitatory cells. Importantly, inhibitory neurons connect also to each other extensively, following a detailed blueprint, and, indeed, specific forms of disinhibition affect important behavioral functions. Here we discuss a peculiar form of cortical disinhibition: the massive autaptic self-inhibition of parvalbumin-(PV) positive basket cells. Despite being described long ago, autaptic inhibition onto PV basket cells is rarely included in cortical circuit diagrams, perhaps because of its still elusive function. We propose here a potential dual role of autaptic feedback inhibition in temporally coordinating PV basket cells during cortical network activity.

Acetaminophen has been used as an analgesic for more than a hundred years, but its mechanism of action has remained elusive. Recently, it has been shown that acetaminophen produces analgesia by the activation of the brain endocannabinoid receptor CB1 through its para-aminophenol (p-aminophenol) metabolite. The objective of this study was to determine whether p-aminophenol could be toxic for in vitro developing mouse corticalneurons as a first step in establishing a link between acetaminophen use and neuronal apoptosis. We exposed developing mouse corticalneurons to various concentrations of drugs for 24 hr in vitro. Acetaminophen itself was not toxic to developing mouse corticalneurons at therapeutic concentrations of 10-250 μg/ml. However, concentrations of p-aminophenol from 1 to 100 μg/ml produced significant (p < 0.05) loss of mouse corticalneuron viability at 24 hr compared to the controls. The naturally occurring endocannabinoid anandamide also caused similar 24-hr loss of cell viability in developing mouse corticalneurons at concentrations from 1 to 100 μg/ml, which indicates the mechanism of cell death could be through the cannabinoid receptors. The results of our experiments have shown a detrimental effect of the acetaminophen metabolite p-aminophenol on in vitro developing corticalneuron viability which could act through CB1 receptors of the endocannabinoid system. These results could be especially important in recommending an analgesic for children or individuals with traumatic brain injury who have developing corticalneurons.

Background Acid–base imbalance in various metabolic disturbances leads to human brain dysfunction. Compared with acidosis, the patients suffered from alkalosis demonstrate more severe neurological signs that are difficultly corrected. We hypothesize a causative process that the nerve cells in the brain are more vulnerable to alkalosis than acidosis. Methods The vulnerability of GABAergic neurons to alkalosis versus acidosis was compared by analyzing their functional changes in response to the extracellular high pH and low pH. The neuronal and synaptic functions were recorded by whole-cell recordings in the cortical slices. Results The elevation or attenuation of extracellular pH impaired these GABAergic neurons in terms of their capability to produce spikes, their responsiveness to excitatory synaptic inputs and their outputs via inhibitory synapses. Importantly, the dysfunction of these active properties appeared severer in alkalosis than acidosis. Conclusions The severer impairment of cortical GABAergic neurons in alkalosis patients leads to more critical neural excitotoxicity, so that alkalosis-induced brain dysfunction is difficultly corrected, compared to acidosis. The vulnerability of cortical GABAergic neurons to high pH is likely a basis of severe clinical outcomes in alkalosis versus acidosis. PMID:24314112

Recent evidence suggests that glutamate-induced cytotoxicity contributes to autophagic neuron death and is partially mediated by increased oxidative stress. Salidroside has been demonstrated to have neuroprotective effects in glutamate-induced neuronal damage. The precise mechanism of its regulatory role in neuronal autophagy is, however, poorly understood. This study aimed to probe the effects and mechanisms of salidroside in glutamate-induced autophagy activation in cultured rat corticalneurons. Cell viability assay, Western blotting, coimmunoprecipitation, and small interfering RNA were performed to analyze autophagy activities during glutamate-evoked oxidative injury. We found that salidroside protected neonatal neurons from glutamate-induced apoptotic cell death. Salidroside significantly attenuated the LC3-II/LC3-I ratio and expression of Beclin-1, but increased (SQSTM1)/p62 expression under glutamate exposure. Pretreatment with 3-methyladenine (3-MA), an autophagy inhibitor, decreased LC3-II/LC3-I ratio, attenuated glutamate-induced cell injury, and mimicked some of the protective effects of salidroside against glutamate-induced cell injury. Molecular analysis demonstrated that salidroside inhibited corticalneuron autophagy in response to glutamate exposure through p53 signaling by increasing the accumulation of cytoplasmic p53. Salidroside inhibited the glutamate-induced dissociation of the Bcl-2-Beclin-1 complex with minor affects on the PI3K/Akt/mTOR signaling pathways. These data demonstrate that the inhibition of autophagy could be responsible for the neuroprotective effects of salidroside on glutamate-induced neuronal injury.

Full Text Available Neural population equations such as neural mass or field models are widely used to study brain activity on a large scale. However, the relation of these models to the properties of single neurons is unclear. Here we derive an equation for several interacting populations at the mesoscopic scale starting from a microscopic model of randomly connected generalized integrate-and-fire neuron models. Each population consists of 50-2000 neurons of the same type but different populations account for different neuron types. The stochastic population equations that we find reveal how spike-history effects in single-neuron dynamics such as refractoriness and adaptation interact with finite-size fluctuations on the population level. Efficient integration of the stochastic mesoscopic equations reproduces the statistical behavior of the population activities obtained from microscopic simulations of a full spiking neural network model. The theory describes nonlinear emergent dynamics such as finite-size-induced stochastic transitions in multistable networks and synchronization in balanced networks of excitatory and inhibitory neurons. The mesoscopic equations are employed to rapidly integrate a model of a cortical microcircuit consisting of eight neuron types, which allows us to predict spontaneous population activities as well as evoked responses to thalamic input. Our theory establishes a general framework for modeling finite-size neural population dynamics based on single cell and synapse parameters and offers an efficient approach to analyzing cortical circuits and computations.

The mammalian neocortex is composed of various types of neurons that reflect its laminar and area structures. It has been suggested that not only intrinsic but also afferent-derived extrinsic factors are involved in neuronal differentiation during development. However, the role and molecular mechanism of such extrinsic factors are almost unknown. Here, we attempted to identify molecules that are expressed in the thalamus and affect cortical cell development. First, thalamus-specific molecules were sought by comparing gene expression profiles of the developing rat thalamus and cortex using microarrays, and by constructing a thalamus-enriched subtraction cDNA library. A systematic screening by in situ hybridization showed that several genes encoding extracellular molecules were strongly expressed in sensory thalamic nuclei. Exogenous and endogenous protein localization further demonstrated that two extracellular molecules, Neuritin-1 (NRN1) and VGF, were transported to thalamic axon terminals. Application of NRN1 and VGF to dissociated cell culture promoted the dendritic growth. An organotypic slice culture experiment further showed that the number of primary dendrites in multipolar stellate neurons increased in response to NRN1 and VGF, whereas dendritic growth of pyramidal neurons was not promoted. These molecules also increased neuronal survival of multipolar neurons. Taken together, these results suggest that the thalamus-specific molecules NRN1 and VGF play an important role in the dendritic growth and survival of corticalneurons in a cell type-specific manner.

Full Text Available Cortical GABAergic interneurons constitute an extremely diverse population of cells organized in a well-defined topology of precisely interconnected cells. They play a crucial role regulating inhibitory-excitatory balance in brain circuits, gating sensory perception and regulating spike timing to brain oscillations during distinct behaviors. Dysfunctions in the establishment of proper inhibitory circuits have been associated to several brain disorders such as autism, epilepsy and schizophrenia. In the rodent adult cortex, inhibitory neurons are generated during the second gestational week from distinct progenitor lineages located in restricted domains of the ventral telencephalon. However, only recently, studies have revealed some of the mechanisms generating the heterogeneity of neuronal subtypes and their modes of integration in brain networks. Here we will discuss some the events involved in the production of cortical GABAergic neuron diversity with focus on the interaction between intrinsically driven genetic programs and environmental signals during development.

The migration of cortical projection neurons is a multistep process characterized by dynamic cell shape remodeling. The molecular basis of these changes remains elusive, and the present work describes how microRNAs (miRNAs) control neuronal polarization during radial migration. We show that miR-22 and miR-124 are expressed in the cortical wall where they target components of the CoREST/REST transcriptional repressor complex, thereby regulating doublecortin transcription in migrating neurons. This molecular pathway underlies radial migration by promoting dynamic multipolar-bipolar cell conversion at early phases of migration, and later stabilization of cell polarity to support locomotion on radial glia fibers. Thus, our work emphasizes key roles of some miRNAs that control radial migration during cerebral corticogenesis.

In the developing cortex, projection neurons undergo multipolar-bipolar transition, radial-directed migration, and maturation. The contribution of these developmental steps to the structure of the adult cortex is not completely understood. Here, we report that huntingtin (HTT), the protein mutated in Huntington's disease, is enriched in polarizing projection neurons. The depletion of HTT in postmitotic projection neurons leads to the mislocalization of layer-specific neuronal populations in the mouse neocortex. HTT is required for the multipolar-bipolar transition of projection neurons and for the maintenance of their bipolar shape during their radial migration. HTT mediates these effects in vivo through the regulation of RAB11-dependent N-Cadherin trafficking. Importantly, HD pathological HTT alters RAB11-dependent neuronal migration. Finally, we show that the cortical defects resulting from the postmitotic loss of HTT specifically during embryonic development affect neuronal morphology at adulthood. Our data reveal a new HTT-RAB11-N-Cadherin pathway regulating multipolar-bipolar transition with direct implications for mature brain. VIDEO ABSTRACT.

Full Text Available Traumatic brain injury (TBI from a blow to the head is often associated with complex patterns of brain abnormalities that accompany deficits in cognitive and motor function. Previously we reported that a long-term consequence of TBI, induced with a closed-head injury method modelling human car and sporting accidents, is neuronal hyper-excitation in the rat sensory barrel cortex that receives tactile input from the face whiskers. Hyper-excitation occurred only in supra-granular layers and was stronger to complex than simple stimuli. We now examine changes in the immediate aftermath of TBI induced with same injury method. At 24 hours post-trauma significant sensorimotor deficits were observed and characterisation of the cortical population neuronal responses at that time revealed a depth-dependent suppression of neuronal responses, with reduced responses from supragranular layers through to input layer IV, but not in infragranular layers. In addition, increased spontaneous firing rate was recorded in cortical layers IV and V. We postulate that this early post-injury suppression of cortical processing of sensory input accounts for immediate post-trauma sensory morbidity and sets into train events that resolve into long-term cortical hyper-excitability in upper sensory cortex layers that may account for long-term sensory hyper-sensitivity in humans with TBI.

The brain-derived neurotrophic factor (BDNF) participates in the regulation of corticalneurons by influencing the release of glutamate. However, the specific mechanisms are unclear. Hence, we isolated and cultured the corticalneurons of Sprague Dawley rats. Specific inhibitors of TrkB, Src, PLC-γ1, Akt, and MEK1/2 (i.e., K252a, PP2, U73122, LY294002, and PD98059, respectively) were used to treat corticalneurons and to detect the glutamate release from corticalneurons stimulated with BDNF. BDNF significantly increased glutamate release, and simultaneously enhanced phosphorylation levels of TrkB, Src, PLC-γ, Akt, and Erk1/2. For BDNF-stimulated corticalneurons, K252a inhibited glutamate release and inhibited the phosphorylation levels of TrkB, Src, PLC-γ, Erk1/2, and Akt (P PLC-γ1 (P 0.05). U73122 inhibited the glutamate release from BDNF-stimulated corticalneurons, but had no influence on the phosphorylation levels of TrkB, Src, Erk1/2, or Akt (P > 0.05). LY294002 and PD98059 did not affect the BDNF-stimulated glutamate release and did not inhibit the phosphorylation levels of TrkB, Src, or PLC-γ1. In summary, BDNF stimulated the glutamate release from corticalneurons via the TrkB/Src/PLC-γ1 signaling pathway.

Brain-derived neurotrophic factor (BDNF) promotes the biochemical and morphological differentiation of selective populations of neurons during development. In this study we examined the energy requirements associated with the effects of BDNF on neuronal differentiation. Because glucose is the preferred energy substrate in the brain, the effect of BDNF on glucose utilization was investigated in developing corticalneurons via biochemical and imaging studies. Results revealed that BDNF increases glucose utilization and the expression of the neuronal glucose transporter GLUT3. Stimulation of glucose utilization by BDNF was shown to result from the activation of Na+/K+-ATPase via an increase in Na+ influx that is mediated, at least in part, by the stimulation of Na+-dependent amino acid transport. The increased Na+-dependent amino acid uptake by BDNF is followed by an enhancement of overall protein synthesis associated with the differentiation of corticalneurons. Together, these data demonstrate the ability of BDNF to stimulate glucose utilization in response to an enhanced energy demand resulting from increases in amino acid uptake and protein synthesis associated with the promotion of neuronal differentiation by BDNF.

Full Text Available Striatal neurons can be either projection neurons or interneurons, with each type exhibiting distinct susceptibility to various types of brain damage. In this study, 6-hydroxydopamine was injected into the right medial forebrain bundle to induce dopamine depletion, and/or ibotenic acid was injected into the M1 cortex to induce motor cortex lesions. Immunohistochemistry and western blot assay showed that dopaminergic depletion results in significant loss of striatal projection neurons marked by dopamine- and cyclic adenosine monophosphate-regulated phosphoprotein, molecular weight 32 kDa, calbindin, and μ-opioid receptor, while cortical lesions reversed these pathological changes. After dopaminergic deletion, the number of neuropeptide Y-positive striatal interneurons markedly increased, which was also inhibited by cortical lesioning. No noticeable change in the number of parvalbumin-positive interneurons was found in 6-hydroxydopamine-treated rats. Striatal projection neurons and interneurons show different susceptibility to dopaminergic depletion. Further, cortical lesions inhibit striatal dysfunction and damage induced by 6-hydroxydopamine, which provides a new possibility for clinical treatment of Parkinson's disease.

Decisions based on sensory evaluation during single trials may depend on the collective activity of neurons distributed across brain circuits. Previous studies have deepened our understanding of how the activity of individual neurons relates to the formation of a decision and its storage for later report. However, little is known about how decision-making and decision maintenance processes evolve in single trials. We addressed this problem by studying the activity of simultaneously recorded neurons from different somatosensory and frontal lobe cortices of monkeys performing a vibrotactile discrimination task. We used the hidden Markov model to describe the spatiotemporal pattern of activity in single trials as a sequence of firing rate states. We show that the animal's decision was reliably maintained in frontal lobe activity through a selective state sequence, initiated by an abrupt state transition, during which many neurons changed their activity in a concomitant way, and for which both latency and variability depended on task difficulty. Indeed, transitions were more delayed and more variable for difficult trials compared with easy trials. In contrast, state sequences in somatosensory cortices were weakly decision related, had less variable transitions, and were not affected by the difficulty of the task. In summary, our results suggest that the decision process and its subsequent maintenance are dynamically linked by a cascade of transient events in frontal lobe cortices.

Arsenic trioxide (As2O3) is very effective for treatment of acute promyelocytic leukaemia (APL) but little can pass through the blood-brain-barrier (BBB),which limits its use in the prevention and treatment of central nervous system leukaemia (CNSL). Before creating a non-invasive method to help As2O3 's access, the safe and effective therapeutic concentration of As2O3 in the CNS ought to be known. The changes of apoptosis biomarkers, [Ca2+]i and PKC activity of both leukaemia cells and human corticalneurons, were monitored before and after being treated with As2O3 in vitro with laser confocal microscopy and Western blot. NSE concentration, the neuron invasive biomarker, was monitored by enzyme immunoassay (NSE-EIA). This study revealed that corticalneuron was more tolerable to As2O3 compared to NB4. 1.0 μmol / L As2O3 showed little influence on corticalneuron but effectively promoted apoptosis and induced differentiation of NB4.

Altered gravity forces might influence neuroplasticity and can provoke changes in biochemical mechanisms. In this contest, neurotrophins have a pivotal role, particularly nerve growth factor (NGF) and brain derived neurotrophic factor (BDNF). A suspension of dissociated cortical cells from rat embryos was exposed to 24 h of microgravity before plating in normal adherent culture system. Expression and transductional signalling pathways of NGF and BDNF were assessed at the end of maturational process (8-10 days in vitro). Rotating wall vessel bioreactor (RWV) pre-exposition did not induce changes in NGF expression and its high affinity receptor TrkA. On the contrary both BDNF expression and its high affinity receptor TrkB were strongly up-regulated, inducing Erk-5, but not Erk-1/2 activation and, in turn, MEF2C over-expression and activation. According to our previous and present results, we postulate that relatively short microgravitational stimuli, applied to neural cells during the developmental stage, exert a long time activation of specific neurotrophic pathways.

To investigate the minimum neuron and neurite densities required for synchronized bursts, we cultured rat corticalneurons on planar multi-electrode arrays (MEAs) at five plating densities (2500, 1000, 500, 250, and 100 cells/mm(2)) using two culture media: Neuron Culture Medium and Dulbecco's Modified Eagle Medium supplemented with serum (DMEM/serum). Long-term recording of spontaneous electrical activity clarified that the cultures exhibiting synchronized bursts required an initial plating density of at least 250 cells/mm(2) for Neuron Culture Medium and 500 cells/mm(2) for DMEM/serum. Immediately after electrical recording, immunocytochemistry of microtubule-associated protein 2 (MAP2) and Neurofilament 200 kD (NF200) was performed directly on MEAs to investigate the actual densities of neurons and neurites forming the networks. Immunofluorescence observation revealed that the construction of complicated neuronal networks required the same initial plating density as for synchronized bursts, and that overly sparse cultures showed significant decreases of neurons and neurites. We also found that the final densities of surviving neurons at 1 month decreased greatly compared with the initial plating densities and became saturated in denser cultures. In addition, the area of neurites and the number of nuclei were saturated in denser cultures. By comparing both the results of electrophysiological recording and immunocytochemical observation, we revealed that there is a minimum threshold of neuron densities that must be met for the exhibition of synchronized bursts. Interestingly, these minimum densities of MAP2-positive final neurons did not differ between the two culture media; the density was approximately 50 neurons/mm(2). This value was obtained in the cultures with the initial plating densities of 250 cells/mm(2) for Neuron Culture Medium and 500 cells/mm(2) for DMEM/serum.

Deficiencies in fragile X mental retardation protein (FMRP) are the most common cause of inherited intellectual disability, fragile X syndrome (FXS), with symptoms manifesting during infancy and early childhood. Using a mouse model for FXS, we found that Fmrp regulates the positioning of neurons in the cortical plate during embryonic development, affecting their multipolar-to-bipolar transition (MBT). We identified N-cadherin, which is crucial for MBT, as an Fmrp-regulated target in embryonic brain. Furthermore, spontaneous network activity and high-resolution brain imaging revealed defects in the establishment of neuronal networks at very early developmental stages, further confirmed by an unbalanced excitatory and inhibitory network. Finally, reintroduction of Fmrp or N-cadherin in the embryo normalized early postnatal neuron activity. Our findings highlight the critical role of Fmrp in the developing cerebral cortex and might explain some of the clinical features observed in patients with FXS, such as alterations in synaptic communication and neuronal network connectivity.

Under pathological conditions such as brain trauma, subarachnoid hemorrhage and stroke, cortical spreading depression (CSD) or peri-infarct depolarizations contribute to brain damage in animal models of neurological disorders as well as in human neurological diseases. CSD causes transient megachannel opening on the neuronal membrane, which may compromise neuronal survival under pathological conditions. Poloxamer-188 (P-188) and citicoline are neuroprotectants with membrane sealing properties. The aim of this study is to investigate the effect of P-188 and citicoline on the neuronal megachannel opening induced by CSD in the mouse brain. We have monitored megachannel opening with propidium iodide, a membrane impermeable fluorescent dye and, demonstrate that P-188 and citicoline strikingly decreased CSD-induced neuronal PI influx in cortex and hippocampal dentate gyrus. Therefore, these agents may be providing neuroprotection by blocking megachannel opening, which may be related to their membrane sealing action and warrant further investigation for treatment of traumatic brain injury and ischemic stroke.

Severe brain injuries can trigger epileptogenesis, a latent period that eventually leads to epilepsy. Previous studies have demonstrated that changes in local connectivity between corticalneurons are a part of the epileptogenic processes. In the present study we aimed to investigate whether changes in long-range connectivity are also involved in epileptogenesis. We performed a large unilateral transection (undercut) of the white matter below the suprasylvian gyrus in cats. After about 2 months, we either injected retrograde tracer (cholera toxin, sub-unit B, CTB) or performed Golgi staining. We analyzed distribution of retrogradely labeled neurons, counted dendritic spines in the neocortex (Golgi staining), and analyzed dendritic orientation in control conditions and after the injury. We found a significant increase in the number of detected cells at the frontal parts of the injured hemisphere, which suggests that the process of axonal sprouting occurs in the deafferented area. The increase in the number of retrogradely stained neurons was accompanied with a significant decrease in neocortical spine density in the undercut area, a reduction in vertical and an increase in horizontal orientation of neuronal processes. The present study shows global morphological changes underlying epileptogenesis. An increased connectivity in the injured cortical regions accompanied with a decrease in spine density suggests that excitatory synapses might be formed on dendritic shafts, which probably contributes to the altered neuronal excitability that was described in previous studies on epileptogenesis.

Calcium imaging is a common technique that is useful for measuring calcium signals in cultured cells. Calcium imaging techniques take advantage of calcium indicator dyes, which are BAPTA-based organic molecules that change their spectral properties in response to the binding of Ca2+ ions. Calcium indicator dyes fall into two categories, ratio-metric dyes like Fura-2 and Indo-1 and single-wavelength dyes like Fluo-4. Ratio-metric dyes change either their excitation or their emission spectra in response to calcium, allowing the concentration of intracellular calcium to be determined from the ratio of fluorescence emission or excitation at distinct wavelengths. The main advantage of using ratio-metric dyes over single wavelength probes is that the ratio signal is independent of the dye concentration, illumination intensity, and optical path length allowing the concentration of intracellular calcium to be determined independently of these artifacts. One of the most common calcium indicators is Fura-2, which has an emission peak at 505 nM and changes its excitation peak from 340 nm to 380 nm in response to calcium binding. Here we describe the use of Fura-2 to measure intracellular calcium elevations in neurons and other excitable cells.

Full Text Available We measured the action potential backpropagation delays in apical dendrites of layer 5 pyramidal neurons of the somatosensory cortex under different stimulation regimes that exclude synaptic involvement. These delays showed robust features and did not correlate to either transient change in the stimulus strength or low frequency stimulation of suprathreshold membrane oscillations. However, our results indicate that backpropagation delays correlate with high frequency (>10 Hz stimulation of membrane oscillations, and that persistent suprathreshold sinusoidal stimulation injected directly into the soma results in an increase of the backpropagation delay, suggesting an intrinsic adaptation of the bAP, which does not involve any synaptic modifications. Moreover, the calcium chelator BAPTA eliminated the alterations in the backpropagation delays, strengthening the hypothesis that increased calcium concentration in the dendrites modulates dendritic excitability and can impact the backpropagation velocity. These results emphasize the impact of dendritic excitability on bAP velocity along the dendritic tree, which affects the precision of the bAP arrival at the synapse during specific stimulus regimes, and is capable of shifting the extent and polarity of synaptic strength during suprathreshold synaptic processes such as STDP.

In summary, our data suggest that the cytotoxic effect of 10 μM Cramb816 in corticalneurons may be related to an increase in the cytosolic calcium concentration elicited by the toxin, which is shown to be mediated by glutamate receptor activation. Further studies analyzing the effect of glutamate receptor blockers on the cytotoxic effect of Cramb816 are needed to confirm this hypothesis.

Full Text Available In the present work, we have studied whether cell death could be induced in corticalneurons from rats subjected to different period of O2 deprivation and low glucose (ODLG. This “in vitro” model is designed to emulate the penumbra area under ischemia. In these conditions, corticalneurons displayed loss of mitochondrial respiratory ability however, nor necrosis neither apoptosis occurred despite ROS production. The absence of cellular death could be a consequence of increased antioxidant responses such as superoxide dismutase-1 (SOD1 and GPX3. In addition, the levels of reduced glutathione were augmented and HIF-1/3α overexpressed. After long periods of ODLG (12–24 h corticalneurons showed cellular and mitochondrial membrane alterations and did not recuperate cellular viability during reperfusion. This could mean that therapies directed toward prevention of cellular and mitochondrial membrane imbalance or cell death through mechanisms other than necrosis or apoptosis, like authophagy, may be a way to prevent ODLG damage.

The link between cell death and increased cyclooxygenases-2 (COX-2) activity has not been clearly established. In this study, we examined whether COX-2 activation contributed to the mechanism of neurotoxicity produced by an organophosphorous nerve agent in cultured rat corticalneurons. Exposure of neuronal cells to the nerve agent, VX resulted in an increase in COX enzyme activity in the culture media. A concentration dependent increase in the activity levels of COX-2 enzyme was observed while there was little to no effect on COX-1. In addition, COX-2 mRNA and protein levels increased several hours post-VX exposure. Pre-treatment of the cortical cells with the COX-2 selective inhibitor, NS 398 resulted in a decrease in both the enzyme activity and prostaglandin (PGE(2) and PGF(2α)) release, as well as in a reduction in cell death. These findings indicate that the increase in COX-2 activity may contribute to the mechanism of VX-induced neurotoxicity in cultured rat corticalneuron.

Full Text Available Medium spiny neurons (MSNs are the major striatal neuron and receive synaptic input from both glutamatergic and dopaminergic afferents. These synapses are made on MSN dendritic spines, which undergo density and morphology changes in association with numerous disease and experience-dependent states. Despite wide interest in the structure and function of mature MSNs, relatively little is known about MSN development. Furthermore, most in vitro studies of MSN development have been done in simple striatal cultures that lack any type of non-autologous synaptic input, leaving open the question of how MSN development is affected by a complex environment that includes other types of neurons, glia, and accompanying secreted and cell-associated cues. Here we characterize the development of MSNs in striatal-cortical co-culture, including quantitative morphological analysis of dendritic arborization and spine development, describing progressive changes in density and morphology of developing spines. Overall, MSN growth is much more robust in the striatal-cortical co-culture compared to striatal mono-culture. Inclusion of dopamine in the co-culture further enhances MSN dendritic arborization and spine density, but the effects of dopamine on dendritic branching are only significant at later times in development. In contrast, exogenous Brain Derived Neurotrophic Factor (BDNF has only a minimal effect on MSN development in the co-culture, but significantly enhances MSN dendritic arborization in striatal mono-culture. Importantly, inhibition of NMDA receptors in the co-culture significantly enhances the effect of exogenous BDNF, suggesting that the efficacy of BDNF depends on the cellular environment. Combined, these studies identify specific periods of MSN development that may be particularly sensitive to perturbation by external factors and demonstrate the importance of studying MSN development in a complex signaling environment.

In spite of recent progress in brain sciences, the local circuit of the cerebral neocortex, including motor areas, still remains elusive. Morphological works on excitatory cortical circuitry from thalamocortical (TC) afferents to corticospinal neurons (CSNs) in motor-associated areas are reviewed here. First, TC axons of motor thalamic nuclei have been re-examined by the single-neuron labeling method. There are middle layer (ML)-targeting and layer (L) 1-preferring TC axon types in motor-associated areas, being analogous to core and matrix types, respectively, of Jones (1998) in sensory areas. However, the arborization of core-like motor TC axons spreads widely and disregards the columnar structure that is the basis of information processing in sensory areas, suggesting that motor areas adopt a different information-processing framework such as area-wide laminar organization. Second, L5 CSNs receive local excitatory inputs not only from L2/3 pyramidal neurons but also from ML spiny neurons, the latter directly processing cerebellar information of core-like TC neurons (TCNs). In contrast, basal ganglia information is targeted to apical dendrites of L2/3 and L5 pyramidal neurons through matrix TCNs. Third, L6 corticothalamic neurons (CTNs) are most densely innervated by ML spiny neurons located just above CTNs. Since CTNs receive only weak connections from L2/3 and L5 pyramidal neurons, the TC recurrent circuit composed of TCNs, ML spiny neurons and CTNs appears relatively independent of the results of processing in L2/3 and L5. It is proposed that two circuits sharing the same TC projection and ML neurons are embedded in the neocortex: one includes L2/3 and L5 neurons, processes afferent information in a feedforward way and sends the processed information to other cortical areas and subcortical regions; and the other circuit participates in a dynamical system of the TC recurrent circuit and may serve as the basis of autonomous activity of the neocortex. PMID

Spinal fixation in the osteoporotic patient can be challenging due to the poor trabecular bone quality of the vertebral body. Patients with osteoporotic vertebral body compression fractures are at risk for future compression fractures at adjacent levels, especially after cement augmentation. The purpose of this technical report is to describe the utilization of a cortical screw trajectory along with kyphoplasty for a patient with an osteoporotic compression fracture as well as degenerative spinal disease. This trajectory allows for the possibility of percutaneous pedicle access in the event of future compression fractures. Our patient underwent a decompressive laminectomy and kyphoplasty at the level of an osteoporotic compression fracture. The fracture was stabilized with cortical screw instrumentation and fusion at a level above and a level below the fracture. Subsequently the patient developed an adjacent level fracture within the fusion construct. Due to the utilization of a cortical screw trajectory for the initial fusion, the traditional pedicle trajectory was still accessible. As a result, the new fracture was treated with a percutaneous kyphoplasty through a standard pedicle trajectory. In conclusion, the use of a cortical screw trajectory for stabilization of osteoporotic compression fractures provides for a stronger bone screw interface and avoids osteoporotic trabecular vertebral body bone. At the same time this trajectory allows for future percutaneous pedicular access in the event that the patient suffers future compression fractures.

While functional recovery after injury is limited, it has become evident that the mature central nervous system does retain some ability to regenerate. This study investigated the intrinsic capacity of relatively mature corticalneurons (21 days in vitro) to respond to axonal loss. Neurons, growing as clusters on poly-L-lysine, were completely sheared of axons through chemical and mechanical disruption and transferred to either an intact astrocyte monolayer or a substrate of poly-L-lysine. Injured neurons exhibited a regenerative sprouting response that was independent of neuronal cell division or neural progenitors, as demonstrated by negative bromodeoxyuridine (BrdU) and the neuronal precursor intermediate filament nestin, labeling. At 24 h after injury, neurons had extended appropriately polarized neurites, demonstrated by compartmentalized microtubule-associated proteins MAP2 and tau immunolabeling. Newly sprouting axons were tipped by growth cones; however, growth cones on the tips of sprouting axons (mean area, 26.32 +/- 2.20 microm) were significantly (pregenerating neurons exhibited distinct axonal dynamics, with a significant (pneuronal structural plasticity and defining the role of astrocyte reactivity in the response to trauma.

Ciguatoxins are sodium channels activators that cause ciguatera, one of the most widespread nonbacterial forms of food poisoning, which presents with long-term neurological alterations. In central neurons, chronic perturbations in activity induce homeostatic synaptic mechanisms that adjust the strength of excitatory synapses and modulate glutamate receptor expression in order to stabilize the overall activity. Immediate early genes, such as Arc and Egr1, are induced in response to activity changes and underlie the trafficking of glutamate receptors during neuronal homeostasis. To better understand the long lasting neurological consequences of ciguatera, it is important to establish the role that chronic changes in activity produced by ciguatoxins represent to central neurons. Here, the effect of a 30 min exposure of 10-13 days in vitro (DIV) corticalneurons to the synthetic ciguatoxin CTX 3C on Arc and Egr1 expression was evaluated using real-time polymerase chain reaction approaches. Since the toxin increased the mRNA levels of both Arc and Egr1, the effect of CTX 3C in NaV channels, membrane potential, firing activity, miniature excitatory postsynaptic currents (mEPSCs), and glutamate receptors expression in corticalneurons after a 24 h exposure was evaluated using electrophysiological and western blot approaches. The data presented here show that CTX 3C induced an upregulation of Arc and Egr1 that was prevented by previous coincubation of the neurons with the NaV channel blocker tetrodotoxin. In addition, chronic CTX 3C caused a concentration-dependent shift in the activation voltage of NaV channels to more negative potentials and produced membrane potential depolarization. Moreover, 24 h treatment of corticalneurons with 5 nM CTX 3C decreased neuronal firing and induced synaptic scaling mechanisms, as evidenced by a decrease in the amplitude of mEPSCs and downregulation in the protein level of glutamate receptors that was also prevented by tetrodotoxin

Full Text Available We are interested in identifying and characterizing various projection neurons that constitute the neocortical circuit. For this purpose, we developed a novel lentiviral vector that carries the tetracycline transactivator (tTA and the transgene under the TET Responsive Element promoter (TRE on a single backbone. By pseudotyping such a vector with modified rabies G-protein, we were able to express palmitoylated-GFP (palGFP or turboFP635 (RFP in corticothalamic, corticocortical, and corticopontine neurons of mice. The high-level expression of the transgene achieved by the TET-Off system enabled us to observe characteristic elaboration of neuronal processes for each cell type. At higher magnification, we were able to observe fine structures such as boutons and spines as well. We also injected our retrograde TET-Off vector to the marmoset cortex and proved that it can be used to label the long-distance cortical connectivity of millimeter scale. In conclusion, our novel retrograde tracer provides an attractive option to investigate the morphologies of identified cortical projection neurons of various species.

We have used a multiunit electrode array in extracellular recording to investigate changes in the firing patterns in networks of developing rat corticalneurons. The spontaneous activity of continual asynchronous firing or the alternation of asynchronous spikes and synchronous bursts changed over time so that activity in the later stages consisted exclusively of synchronized bursts. The spontaneous coordinated activity in bursts produced a variability in interburst interval (IBI) sequences that is referred to as ``form.'' The stochastic and nonlinear dynamical analysis of IBI sequences revealed that these sequences reflected a largely random process and that the form for relatively immature neurons was largely oscillatory while the form for the more mature neurons was Poisson-like. The observed IBI sequences thus showed changes in form associated with both the intrinsic properties of the developing cells and the neural response to correlated synaptic inputs due to interaction between the developing neural circuits.

BACKGROUND: Cholecystokinin (CCK-8) can regulate the synthesis of NO, release of amino acid substance and suppress Ca2+ inflow. It is unknown about neuroprotection of CCK-8 on neuronal apoptosis and its relationship with nerve growth factor (NGF).OBJECTryE: To investigate the protective effect of CCK-8 on in vitro cultured rat corticalneurons against apoptosis induced by glutamate, and explore its effect on expression of NGF in the neurons during apoptosis.DESIGN: Randomized controlled experiment on the basis of cells.SETTING: Children's Research Institute Affiliated to Children Hospital of Chongqing Medical University.MATERIALS: Eighty SD rats of 1-day old; DMEM/F12 culture medium (Biochrom Company, Germany);Fetal bovine serum (TBD Company, Tianjin); CCK-8 (Sigma Company, USA). Glutamate (Bioengineering Company, Shanghai); TUNEL kit and NGF- in situ hybridization kit (Boster Bioengineering Company,Wuhan); anti-NGF polyclonal antibody (Santa-Cluz Company); NGF immunocytochemistry kit (Zhongshan Company, Beijing).METHODS: The experiments were carried out in Children's Research Institute Affiliated to Children Hospital of Chongqing Medical University from December 2004 to September 2005. Primary cultured corticalneurons from SD rats of 1-day oldwere incubated for 7 days. The cultured cells were divided randomly into 3 groups:experimental group, model group and control group. Neurons in experimental groups were added CCK-8 of 1 ×10-6, 1 ×10-7, 1 ×10-8 μ mol/L respectively, and then added 50 μmol/L glutamate solution a hour later. Neurons in model groups were treated with 50 μ mol/L glutamate solution. In the control group, cells were treated with normal medium. Apoptosis of cultured corticalneurons were observed by fluorescent microscope, the expression of NGF protein and mRNA were determined respectively by immunocytochemistry and in situ hybridization, and apoptosis of corticalneurons was detected with terminal deoxynucleotidyl transferase-mediated nick

Repetitive Transcranial Magnetic Stimulation (rTMS) has been successfully used as a non-invasive therapeutic intervention for several neurological disorders in the clinic as well as an investigative tool for basic neuroscience. rTMS has been shown to induce long-term changes in neuronal circuits in vivo. Such long-term effects of rTMS have been investigated using behavioral, imaging, electrophysiological, and molecular approaches, but there is limited understanding of the immediate effects of TMS on neurons. We investigated the immediate effects of high frequency (20 Hz) rTMS on the activity of corticalneurons in an effort to understand the underlying cellular mechanisms activated by rTMS. We used whole-cell patch-clamp recordings in acute rat brain slices and calcium imaging of cultured primary neurons to examine changes in neuronal activity and intracellular calcium respectively. Our results indicate that each TMS pulse caused an immediate and transient activation of voltage gated sodium channels (9.6 ± 1.8 nA at -45 mV, p value rTMS stimulation induced action potentials in a subpopulation of neurons, and significantly increased the steady state current of the neurons at near threshold voltages (at -45 mV: before TMS: I = 130 ± 17 pA, during TMS: I = 215 ± 23 pA, p value = 0.001). rTMS stimulation also led to a delayed increase in intracellular calcium (153.88 ± 61.94% increase from baseline). These results show that rTMS has an immediate and cumulative effect on neuronal activity and intracellular calcium levels, and suggest that rTMS may enhance neuronal responses when combined with an additional motor, sensory or cognitive stimulus. Thus, these results could be translated to optimize rTMS protocols for clinical as well as basic science applications. PMID:28114421

Background Thrombin’s role in the nervous system is not well understood. Under conditions of blood-brain barrier compromise (e.g., neurosurgery or stroke), thrombin can result in neuroapoptosis and the formation of glial scars. Despite this, preconditioning with thrombin has been found to be neuroprotective in models of cerebral ischemia and intracerebral hemorrhage. Methods We investigated the effects of physiologically relevant concentrations of thrombin on corticalneurons using two culture-based assays. We examined thrombin’s effect on neurites by quantitative analysis of fluorescently labeled neurons. To characterize thrombin’s effects on neuron survival, we spectrophotometrically measured changes in enzymatic activity. Using receptor agonists and thrombin inhibitors, we separately examined the role of thrombin and its receptor in neuroprotection. Results We found that low concentrations of thrombin (1 nM) enhances neurite growth and branching, neuron viability, and protects against excitotoxic damage. In contrast, higher concentrations of thrombin (100 nM) are potentially detrimental to neuronal health as evidenced by inhibition of neurite growth. Lower concentrations of thrombin resulted in equivalent neuroprotection as the antifibrinolytic, aprotinin, and the direct thrombin inhibitor, argatroban. Interestingly, exogenous application of the species-specific thrombin inhibitor, antithrombin III, was detrimental to neuronal health; suggesting that some endogenous thrombin is necessary for optimal neuron health in our culture system. Activation of the thrombin receptor, protease-activated receptor - 1 (PAR-1), via micromolar concentrations of the thrombin receptor agonist peptide, TRAP, did not adversely affect neuronal viability. Conclusions An optimal concentration of thrombin exists to enhance neuronal health. Neurotoxic effects of thrombin do not involve activation of PAR receptors and thus separate pharmacologic manipulation of thrombin’s receptor

Full Text Available Elevated homocysteine (Hcy levels have been reported to be involved in neurotoxicity after ischemic stroke. However, the underlying mechanisms remain incompletely understood to date. In the current study, we hypothesized that neuronal autophagy activation may be involved in the toxic effect of Hcy on corticalneurons following cerebral ischemia. Brain cell injury was determined by hematoxylin-eosin (HE staining and TdT-mediated dUTP Nick-End Labeling (TUNEL staining. The level and localization of autophagy were detected by transmission electron microscopy, western blot and immunofluorescence double labeling. The oxidative DNA damage was revealed by immunofluorescence of 8-Hydroxy-2′-deoxyguanosine (8-OHdG. Hcy treatment aggravated neuronal cell death, significantly increased the formation of autophagosomes and the expression of LC3B and Beclin-1 in the brain cortex after middle cerebral artery occlusion-reperfusion (MCAO. Immunofluorescence analysis of LC3B and Beclin-1 distribution indicated that their expression occurred mainly in neurons (NeuN-positive and hardly in astrocytes (GFAP-positive. 8-OHdG expression was also increased in the ischemic cortex of Hcy-treated animals. Conversely, LC3B and Beclin-1 overexpression and autophagosome accumulation caused by Hcy were partially blocked by the autophagy inhibitor 3-methyladenine (3-MA. Hcy administration enhanced neuronal autophagy, which contributes to cell death following cerebral ischemia. The oxidative damage-mediated autophagy may be a molecular mechanism underlying neuronal cell toxicity of elevated Hcy level.

In collaboration with Marshall Nirenberg, we performed in vivo RNA interference (RNAi) genome-wide screening in Drosophila embryos. Pebble has been shown to be involved in Drosophila neuronal development. We have also reported that depletion of Ect2, a mammalian ortholog of Pebble, induces differentiation in NG108-15 neuronal cells. However, the precise role of Ect2 in neuronal development has yet to be studied. Here, we confirmed in PC12 pheochromocytoma cells that inhibition of Ect2 expression by RNAi stimulated neurite outgrowth, and in the mouse embryonic cortex that Ect2 was accumulated throughout the ventricular and subventricular zones with neuronal progenitor cells. Next, the effects of Ect2 depletion were studied in primary cultures of mouse embryonic corticalneurons: Loss of Ect2 did not affect the differentiation stages of neuritogenesis, the number of neurites, or axon length, while the numbers of growth cones and growth cone-like structures were increased. Taken together, our results suggest that Ect2 contributes to neuronal morphological differentiation through regulation of growth cone dynamics. PMID:22366651

Full Text Available Lactate is increasingly described as an energy substrate of the brain. Beside this still debated metabolic role, lactate may have other effects on brain cells. Here, we describe lactate as a neuromodulator, able to influence the activity of corticalneurons. Neuronal excitability of mouse primary neurons was monitored by calcium imaging. When applied in conjunction with glucose, lactate induced a decrease in the spontaneous calcium spiking frequency of neurons. The effect was reversible and concentration dependent (IC50 ∼4.2 mM. To test whether lactate effects are dependent on energy metabolism, we applied the closely related substrate pyruvate (5 mM or switched to different glucose concentrations (0.5 or 10 mM. None of these conditions reproduced the effect of lactate. Recently, a Gi protein-coupled receptor for lactate called HCA1 has been introduced. To test if this receptor is implicated in the observed lactate sensitivity, we incubated cells with pertussis toxin (PTX an inhibitor of Gi-protein. PTX prevented the decrease of neuronal activity by L-lactate. Moreover 3,5-dyhydroxybenzoic acid, a specific agonist of the HCA1 receptor, mimicked the action of lactate. This study indicates that lactate operates a negative feedback on neuronal activity by a receptor-mediated mechanism, independent from its intracellular metabolism.

Lactate is increasingly described as an energy substrate of the brain. Beside this still debated metabolic role, lactate may have other effects on brain cells. Here, we describe lactate as a neuromodulator, able to influence the activity of corticalneurons. Neuronal excitability of mouse primary neurons was monitored by calcium imaging. When applied in conjunction with glucose, lactate induced a decrease in the spontaneous calcium spiking frequency of neurons. The effect was reversible and concentration dependent (IC50 ∼4.2 mM). To test whether lactate effects are dependent on energy metabolism, we applied the closely related substrate pyruvate (5 mM) or switched to different glucose concentrations (0.5 or 10 mM). None of these conditions reproduced the effect of lactate. Recently, a Gi protein-coupled receptor for lactate called HCA1 has been introduced. To test if this receptor is implicated in the observed lactate sensitivity, we incubated cells with pertussis toxin (PTX) an inhibitor of Gi-protein. PTX prevented the decrease of neuronal activity by L-lactate. Moreover 3,5-dyhydroxybenzoic acid, a specific agonist of the HCA1 receptor, mimicked the action of lactate. This study indicates that lactate operates a negative feedback on neuronal activity by a receptor-mediated mechanism, independent from its intracellular metabolism.

Many population-based epidemiological studies have unveiled an inverse correlation between intake of herbal plants and incidence of stroke. C. nutans is a traditional herbal medicine widely used for snake bite, viral infection and cancer in Asian countries. However, its role in protecting stroke damage remains to be studied. Despite of growing evidence to support epigenetic regulation in the pathogenesis and recovery of stroke, a clear understanding of the underlying molecular mechanisms is still lacking. In the present study, primary corticalneurons were subjected to in vitro oxygen-glucose deprivation (OGD)-reoxygenation and hypoxic neuronal death was used to investigate the interaction between C. nutans and histone deacetylases (HDACs). Using pharmacological agents (HDAC inhibitor/activator), loss-of-function (HDAC siRNA) and gain-of-function (HDAC plasmid) approaches, we demonstrated an early induction of HDAC1/2/3/8 and HDAC6 in neurons after OGD insult. C. nutans extract selectively inhibited HDAC1 and HDAC6 expression and attenuated neuronal death. Results of reporter analysis further revealed that C. nutans suppressed HDAC1 and HDAC6 transcription. Besides ameliorating neuronal death, C. nutans also protected astrocytes and endothelial cells from hypoxic-induced cell death. In summary, results support ability for C. nutans to suppress post-hypoxic HDACs activation and mitigate against OGD-induced neuronal death. This study further opens a new avenue for the use of herbal medicines to regulate epigenetic control of brain injury.

Vesicle transport is a major underlying mechanism of cell communication. Inhibiting vesicle transport in brain cells results in blockage of neuronal signals, even in intact neuronal networks. Modulating intracellular vesicle transport can have a huge impact on the development of new neurotherapeutic concepts, but only if we can specifically interfere with intracellular transport patterns. Here, we propose to modulate motion of intracellular lipid vesicles in rat corticalneurons based on exogenously bioconjugated and cell internalized superparamagnetic iron oxide nanoparticles (SPIONs) within microengineered magnetic gradients on-chip. Upon application of 6-126 pN on intracellular vesicles in neuronal cells, we explored how the magnetic force stimulus impacts the motion pattern of vesicles at various intracellular locations without modulating the entire cell morphology. Altering vesicle dynamics was quantified using, mean square displacement, a caging diameter and the total traveled distance. We observed a de-acceleration of intercellular vesicle motility, while applying nanomagnetic forces to cultured neurons with SPIONs, which can be explained by a decrease in motility due to opposing magnetic force direction. Ultimately, using nanomagnetic forces inside neurons may permit us to stop the mis-sorting of intracellular organelles, proteins and cell signals, which have been associated with cellular dysfunction. Furthermore, nanomagnetic force applications will allow us to wirelessly guide axons and dendrites by exogenously using permanent magnetic field gradients.

In collaboration with Marshall Nirenberg, we performed in vivo RNA interference (RNAi) genome-wide screening in Drosophila embryos. Pebble has been shown to be involved in Drosophila neuronal development. We have also reported that depletion of Ect2, a mammalian ortholog of Pebble, induces differentiation in NG108-15 neuronal cells. However, the precise role of Ect2 in neuronal development has yet to be studied. Here, we confirmed in PC12 pheochromocytoma cells that inhibition of Ect2 expression by RNAi stimulated neurite outgrowth, and in the mouse embryonic cortex that Ect2 was accumulated throughout the ventricular and subventricular zones with neuronal progenitor cells. Next, the effects of Ect2 depletion were studied in primary cultures of mouse embryonic corticalneurons: Loss of Ect2 did not affect the differentiation stages of neuritogenesis, the number of neurites, or axon length, while the numbers of growth cones and growth cone-like structures were increased. Taken together, our results suggest that Ect2 contributes to neuronal morphological differentiation through regulation of growth cone dynamics.

Lead exposure has devastating effects on the developing nervous system, producing morphological, cognitive, and behavioral deficits. To elucidate some of the mechanisms of lead neurotoxicity, we have examined its effects on the differentiation of several types of cultured neurons. Previously, we reported the effects of inorganic lead on several parameters of growth and differentiation of E18 rat hippocampal neurons and two types of neuroblastoma cells cultured in medium with 2% fetal calf serum (FCS) (Audesirk et al., 1991). In the present study, we report the effects of concentrations of lead ranging from 1nM to 1 mM on the differentiation of hippocampal neurons cultured in medium containing 10% FCS. In addition, we investigated lead effects on neurons isolated from the motor cortex region of the E18 rat embryo. Corticalneurons were exposed to lead in concentrations ranging from 0.1 nM to 1 mM in medium with either 10% FCS or 2% FCS for 48 hr. The effects of lead tended to be multimodal. Neurite initiation, which is highly sensitive to neurotoxic compounds, was inhibited by lead at both high and low concentrations, with no effects at intermediate levels. Medium with 10% FCS enhanced certain growth parameters and tended to reduce the effects of lead. There was an overall consistency in the effects of lead on motor cortex and hippocampal neurons.

Full Text Available Neocortical cholinergic activity plays a fundamental role in sensory processing and cognitive functions. Previous results have suggested a refined anatomical and functional topographical organization of basal forebrain (BF projections that may control cortical sensory processing in a specific manner. We have used retrograde anatomical procedures to demonstrate the existence of specific neuronal groups in the BF involved in the control of specific sensory cortices. Fluoro-gold and Fast Blue fluorescent retrograde tracers were deposited into the primary somatosensory (S1 and primary auditory (A1 cortices in mice. Our results revealed that the BF is a heterogeneous area in which neurons projecting to different cortical areas are segregated into different neuronal groups. Most of the neurons located in the horizontal limb of the diagonal band of Broca (HDB projected to the S1 cortex, indicating that this area is specialized in the sensory processing of tactile stimuli. However, the nucleus basalis magnocellularis (B nucleus shows a similar number of cells projecting to the S1 as to the A1 cortices. In addition, we analyzed the cholinergic effects on the S1 and A1 cortical sensory responses by optogenetic stimulation of the BF neurons in urethane-anesthetized transgenic mice. We used transgenic mice expressing the light-activated cation channel, channelrhodopsin-2, tagged with a fluorescent protein (ChR2-YFP under the control of the choline-acetyl transferase promoter (ChAT. Cortical evoked potentials were induced by whisker deflections or by auditory clicks. According to the anatomical results, optogenetic HDB stimulation induced more extensive facilitation of tactile evoked potentials in S1 than auditory evoked potentials in A1, while optogenetic stimulation of the B nucleus facilitated either tactile or auditory evoked potentials equally. Consequently, our results suggest that cholinergic projections to the cortex are organized into segregated

Neocortical cholinergic activity plays a fundamental role in sensory processing and cognitive functions. Previous results have suggested a refined anatomical and functional topographical organization of basal forebrain (BF) projections that may control cortical sensory processing in a specific manner. We have used retrograde anatomical procedures to demonstrate the existence of specific neuronal groups in the BF involved in the control of specific sensory cortices. Fluoro-Gold (FlGo) and Fast Blue (FB) fluorescent retrograde tracers were deposited into the primary somatosensory (S1) and primary auditory (A1) cortices in mice. Our results revealed that the BF is a heterogeneous area in which neurons projecting to different cortical areas are segregated into different neuronal groups. Most of the neurons located in the horizontal limb of the diagonal band of Broca (HDB) projected to the S1 cortex, indicating that this area is specialized in the sensory processing of tactile stimuli. However, the nucleus basalis magnocellularis (B) nucleus shows a similar number of cells projecting to the S1 as to the A1 cortices. In addition, we analyzed the cholinergic effects on the S1 and A1 cortical sensory responses by optogenetic stimulation of the BF neurons in urethane-anesthetized transgenic mice. We used transgenic mice expressing the light-activated cation channel, channelrhodopsin-2, tagged with a fluorescent protein (ChR2-YFP) under the control of the choline-acetyl transferase promoter (ChAT). Cortical evoked potentials were induced by whisker deflections or by auditory clicks. According to the anatomical results, optogenetic HDB stimulation induced more extensive facilitation of tactile evoked potentials in S1 than auditory evoked potentials in A1, while optogenetic stimulation of the B nucleus facilitated either tactile or auditory evoked potentials equally. Consequently, our results suggest that cholinergic projections to the cortex are organized into segregated

BACKGROUND:The neuronal transient outward potassium channel has been shown to be highly associated with acetylcholine.However,the influence of acetylcholine on the transient outward potassium current in cerebral corticalneurons remains poorly understood.OBJECTIVE:To investigate acetylcholine modulation on transient outward potassium current in rat parietal corticalneurons using the whole-cell patch-clamp technique.DESIGN,TIME AND SETTING:A neuroelectrophysiology study was performed at the Department of Physiology,Harbin Medical University between January 2005 and January 2006.MATERIALS:Wistar rats were provided by the Animal Research Center,the Second Hospital of Harbin Medical University;PC-IIC patch-clamp amplifier and IBBClamp data collection analysis system were provided by Huazhong University for Science and Technology,Wuhan,China;PP-83 microelectrode puller was purchased from Narrishage,Japan.METHODS:The parietal somatosensory corticalneurons were acutely dissociated,and the modulation of acetylcholine (0.1,1,10,100 μmol/L) on transient outward potassium channel was recorded using the whole-cell patch-clamp technique.MAIN OUTCOME MEASURES:Influence of acetylcholine on transient outward potassium current,potassium channel activation,and inactivation.RESULTS:The inhibitory effect of acetylcholine on transient outward potassium current was dose- and voltage-dependent (P<0.01).Acetylcholine was found to significantly affect the activation process of transient outward potassium current,i.e.,the activation curve of transient outward potassium current was left-shifted,while the inactivation curve was shifted to hyperpolarization.Acetylcholine significantly prolonged the time constant of recovery from inactivation of transient outward potassium current (P<0.01).CONCLUSION:These results suggest that acetylcholine inhibits transient outward potassium current by regulating activation and inactivation processes of the transient outward potassium channel.

An increasing number of studies have demonstrated that insulin-degrading enzyme (IDE) plays an essential role in both the degradation and its activity of β-amyloid (Aβ). Therefore, the regulation of IDE expression and/or modification of IDE-dependent actions are two emerging strategies for the treatment of Alzheimer's disease (AD). We previously observed that geniposide, a novel agonist of glucagon-like peptide 1 receptor (GLP-1R), could attenuate Aβ-induced neurotoxicity by regulating the expression of IDE in primary corticalneurons. However, the signal transduction mechanisms underlying this effect were not elucidated. The present study, therefore examined and explored the cell signaling transduction and molecular mechanisms by which geniposide induces the expression of IDE in primary corticalneurons. The current study revealed that LY294002 (an inhibitor for phosphatidyl inositol 3-kinase, PI3K), PP1 (inhibitor for c-Src), GW9662 (antagonist for peroxisome proliferator-activated receptor γ, PPARγ), H89 (an inhibitor for protein kinase A, PKA) and AG1478 (an antagonist for epidermal growth factor receptor, EGFR) prohibited the up-regulation of IDE induced by geniposide in primary corticalneurons. Further, geniposide also enhanced the phosphorylation of PPARγ and accelerated the release of phosphorylated FoxO1 (forkhead box O1) from nuclear fraction to the cytosol. Moreover, geniposide directly activated the activity of IDE promoter in PC12 cells, which confirmed the presence of the GLP-1 receptor. Taken together, our findings reveal for the first time the cell signaling transduction pathway of geniposide regulating the expression of IDE in neurons.

Full Text Available BACKGROUND: Tissue inhibitor of metalloproteinases-1 (TIMP-1 displays pleiotropic activities, both dependent and independent of its inhibitory activity on matrix metalloproteinases (MMPs. In the central nervous system (CNS, TIMP-1 is strongly upregulated in reactive astrocytes and corticalneurons following excitotoxic/inflammatory stimuli, but no information exists on its effects on growth and morphology of corticalneurons. PRINCIPAL FINDINGS: We found that 24 h incubation with recombinant TIMP-1 induced a 35% reduction in neurite length and significantly increased growth cones size and the number of F-actin rich microprocesses. TIMP-1 mediated reduction in neurite length affected both dendrites and axons after 48 h treatment. The effects on neurite length and morphology were not elicited by a mutated form of TIMP-1 inactive against MMP-1, -2 and -3, and still inhibitory for MMP-9, but were mimicked by a broad spectrum MMP inhibitor. MMP-9 was poorly expressed in developing corticalneurons, unlike MMP-2 which was present in growth cones and whose selective inhibition caused neurite length reductions similar to those induced by TIMP-1. Moreover, TIMP-1 mediated changes in cytoskeleton reorganisation were not accompanied by modifications in the expression levels of actin, betaIII-tubulin, or microtubule assembly regulatory protein MAP2c. Transfection-mediated overexpression of TIMP-1 dramatically reduced neuritic arbour extension in the absence of detectable levels of released extracellular TIMP-1. CONCLUSIONS: Altogether, TIMP-1 emerges as a modulator of neuronal outgrowth and morphology in a paracrine and autrocrine manner through the inhibition, at least in part, of MMP-2 and not MMP-9. These findings may help us understand the role of the MMP/TIMP system in post-lesion pre-scarring conditions.

Cell cycle dysregulation leads to abnormal proliferation and cell death in a context-specific manner. Cell cycle progression driven via the Rb pathway forces neurons to undergo S-phase, resulting in cell death associated with the progression of neuronal degeneration. Nevertheless, some Rb- and Rb family (Rb, p107 and p130)-deficient differentiating neurons can proliferate and form tumors. Here, we found in mouse that differentiating cerebral cortical excitatory neurons underwent S-phase progression but not cell division after acute Rb family inactivation in differentiating neurons. However, the differentiating neurons underwent cell division and proliferated when Rb family members were inactivated in cortical progenitors. Differentiating neurons generated from Rb(-/-); p107(-/-); p130(-/-) (Rb-TKO) progenitors, but not acutely inactivated Rb-TKO differentiating neurons, activated the DNA double-strand break (DSB) repair pathway without increasing trimethylation at lysine 20 of histone H4 (H4K20), which has a role in protection against DNA damage. The activation of the DSB repair pathway was essential for the cell division of Rb-TKO differentiating neurons. These results suggest that newly born corticalneurons from progenitors become epigenetically protected from DNA damage and cell division in an Rb family-dependent manner.

We used RNA interference (RNAi) to disrupt synthesis of the corticalneuronal γ-aminobutyric acid A receptor (GABAAR) α1 in rats during development, and measured outward K+ currents during neuronal electrical activity using whole-cell patch-clamp techniques. Three pairs of small interfering RNA (siRNA) for GABAAR α1 subunit were designed using OligoEngine RNAi software. This siRNA was found to effectively inhibited GABAAR α1 mRNA expression in corticalneuronal culture in vitro, but did not significantly affect neuronal survival. Outward K+ currents were decreased, indicating that GABAAR α1 subunits in developing neurons participate in neuronal function by regulating outward K+ current.

The proximate cause of Parkinson's disease is striatal dopamine depletion. Although no overt toxicity to striatal neurons has been reported in Parkinson's disease, one of the consequences of striatal dopamine loss is a decrease in the number of dendritic spines on striatal medium spiny neurons (MSNs). Dendrites of these neurons receive cortical glutamatergic inputs onto the dendritic spine head and dopaminergic inputs from the substantia nigra onto the spine neck. This synaptic arrangement suggests that dopamine gates corticostriatal glutamatergic drive onto spines. Using triple organotypic slice cultures composed of ventral mesencephalon, striatum, and cortex of the neonatal rat, we examined the role of the cortex in dopamine depletion-induced dendritic spine loss in MSNs. The striatal dopamine innervation was lesioned by treatment of the cultures with the dopaminergic neurotoxin 1-methyl-4-phenylpyridinium (MPP+) or by removing the mesencephalon. Both MPP+ and mesencephalic ablation decreased MSN dendritic spine density. Analysis of spine morphology revealed that thin spines were preferentially lost after dopamine depletion. Removal of the cortex completely prevented dopamine depletion-induced spine loss. These data indicate that the dendritic remodeling of MSNs seen in parkinsonism occurs secondary to increases in corticostriatal glutamatergic drive, and suggest that modulation of cortical activity may be a useful therapeutic strategy in Parkinson's disease.

In this study, we investigated how microtubule motors organize microtubules in Drosophila neurons. We showed that, during the initial stages of axon outgrowth, microtubules display mixed polarity and minus-end-out microtubules push the tip of the axon, consistent with kinesin-1 driving outgrowth by sliding antiparallel microtubules. At later stages, the microtubule orientation in the axon switches from mixed to uniform polarity with plus-end-out. Dynein knockdown prevents this rearrangement and results in microtubules of mixed orientation in axons and accumulation of microtubule minus-ends at axon tips. Microtubule reorganization requires recruitment of dynein to the actin cortex, as actin depolymerization phenocopies dynein depletion, and direct recruitment of dynein to the membrane bypasses the actin requirement. Our results show that cortical dynein slides 'minus-end-out' microtubules from the axon, generating uniform microtubule arrays. We speculate that differences in microtubule orientation between axons and dendrites could be dictated by differential activity of cortical dynein.

In this study, a rat model of transient focal cerebral ischemia was established by performing 100 minutes of middle cerebral artery occlusion, and an in vitro model of experimental oxygen-glucose deprivation using cultured rat corticalneurons was established. Proprotein convertase 2 activity gradually decreased in the ischemic cortex with increasing duration of reperfusion. In cultured rat corticalneurons, the number of terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate-biotin nick end labeling-positive neurons significantly increased and proprotein convertase 2 activity also decreased gradually with increasing duration of oxygen-glucose deprivation. These experimental findings indicate that proprotein convertase 2 activity decreases in ischemic rat cortex after reperfusion, as well as in cultured rat corticalneurons after oxygen-glucose deprivation. These changes in enzyme activity may play an important pathological role in brain injury.

Intermediate progenitors (IPs) amplify the production of pyramidal neurons, but their role in selective genesis of cortical layers or neuronal subtypes remains unclear. Using genetic lineage tracing in mice, we find that IPs destined to produce upper cortical layers first appear early in corticogenesis, by embryonic day 11.5. During later corticogenesis, IP laminar fates are progressively limited to upper layers. We examined the role of Tbr2, an IP-specific transcription factor, in laminar fate regulation using Tbr2 conditional mutant mice. Upon Tbr2 inactivation, fewer neurons were produced by immediate differentiation and laminar fates were shifted upward. Genesis of subventricular mitoses was, however, not reduced in the context of a Tbr2-null cortex. Instead, neuronal and laminar differentiation were disrupted and delayed. Our findings indicate that upper-layer genesis depends on IPs from many stages of corticogenesis and that Tbr2 regulates the tempo of laminar fate implementation for all cortical layers.

Full Text Available Intermediate progenitors (IPs amplify the production of pyramidal neurons, but their role in selective genesis of cortical layers or neuronal subtypes remains unclear. Using genetic lineage tracing in mice, we find that IPs destined to produce upper cortical layers first appear early in corticogenesis, by embryonic day 11.5. During later corticogenesis, IP laminar fates are progressively limited to upper layers. We examined the role of Tbr2, an IP-specific transcription factor, in laminar fate regulation using Tbr2 conditional mutant mice. Upon Tbr2 inactivation, fewer neurons were produced by immediate differentiation and laminar fates were shifted upward. Genesis of subventricular mitoses was, however, not reduced in the context of a Tbr2-null cortex. Instead, neuronal and laminar differentiation were disrupted and delayed. Our findings indicate that upper-layer genesis depends on IPs from many stages of corticogenesis and that Tbr2 regulates the tempo of laminar fate implementation for all cortical layers.

Full Text Available The characteristic six-layered appearance of the neocortex arises from the correct positioning of pyramidal neurons during development and alterations in this process can cause intellectual disabilities and developmental delay. Malformations in cortical development arise when neurons either fail to migrate properly from the germinal zones or fail to cease migration in the correct laminar position within the cortical plate. The Reelin signalling pathway is vital for correct neuronal positioning as loss of Reelin leads to a partially inverted cortex. The precise biological function of Reelin remains controversial and debate surrounds its role as a chemoattractant or stop signal for migrating neurons. To investigate this further we developed an in silico agent-based model of cortical layer formation. Using this model we tested four biologically plausible hypotheses for neuron motility and four biologically plausible hypotheses for the loss of neuron motility (conversion from migration. A matrix of 16 combinations of motility and conversion rules was applied against the known structure of mouse cortical layers in the wild-type cortex, the Reelin-null mutant, the Dab1-null mutant and a conditional Dab1 mutant. Using this approach, many combinations of motility and conversion mechanisms can be rejected. For example, the model does not support Reelin acting as a repelling or as a stopping signal. In contrast, the study lends very strong support to the notion that the glycoprotein Reelin acts as a chemoattractant for neurons. Furthermore, the most viable proposition for the conversion mechanism is one in which conversion is affected by a motile neuron sensing in the near vicinity neurons that have already converted. Therefore, this model helps elucidate the function of Reelin during neuronal migration and cortical development.

Trans-anethole has been studied on pharmacological properties such as anti-inflammation, anti-oxidative stress, antifungal and anticancer. However, to date, the anti-ischemic effects of trans-anethole have not been assessed. Therefore, we investigated the neuroprotection of trans-anethole against oxygen-glucose deprivation/reoxygenation (OGD/R)-induced corticalneuronal cell injury, an in vitro model of ischemia. The abilities of trans-anethole to block excitotoxicity, oxidative stress and mitochondrial dysfunction were evaluated in OGD/R-induced neurons. Trans-anethole significantly ameliorated OGD/R-induced neuronal cell injury by attenuating the intracellular calcium overload via the activation of NMDA receptors. Trans-anethole also inhibited OGD/R-induced reactive oxygen species overproduction, which may be derived from the scavenging activity in peroxyl radicals, assessed in an oxygen radical absorbance capacity assay. Furthermore, trans-anethole was shown to attenuate the depolarization of mitochondrial transmembrane. These results indicated that the neuroprotective effect of trans-anethole on OGD/R-induced neuronal injury might be due to its ability to inhibit excitotoxicity, oxidative stress and mitochondrial dysfunction. Considering these multiple pathways causing ischemic neuronal damage, the multi-functional effect of trans-anethole suggested that it may be effective in treating ischemic stroke.

Full Text Available BACKGROUND: Optogenetic manipulation of a neuronal network enables one to reveal how high-order functions emerge in the central nervous system. One of the Chlamydomonas rhodopsins, channelrhodopsin-1 (ChR1, has several advantages over channelrhodopsin-2 (ChR2 in terms of the photocurrent kinetics. Improved temporal resolution would be expected by the optogenetics using the ChR1 variants with enhanced photocurrents. METHODOLOGY/PRINCIPAL FINDINGS: The photocurrent retardation of ChR1 was overcome by exchanging the sixth helix domain with its counterpart in ChR2 producing Channelrhodopsin-green receiver (ChRGR with further reform of the molecule. When the ChRGR photocurrent was measured from the expressing HEK293 cells under whole-cell patch clamp, it was preferentially activated by green light and has fast kinetics with minimal desensitization. With its kinetic advantages the use of ChRGR would enable one to inject a current into a neuron by the time course as predicted by the intensity of the shedding light (opto-current clamp. The ChRGR was also expressed in the motor corticalneurons of a mouse using Sindbis pseudovirion vectors. When an oscillatory LED light signal was applied sweeping through frequencies, it robustly evoked action potentials synchronized to the oscillatory light at 5-10 Hz in layer 5 pyramidal cells in the cortical slice. The ChRGR-expressing neurons were also driven in vivo with monitoring local field potentials (LFPs and the time-frequency energy distribution of the light-evoked response was investigated using wavelet analysis. The oscillatory light enhanced both the in-phase and out-phase responses of LFP at the preferential frequencies of 5-10 Hz. The spread of activity was evidenced by the fact that there were many c-Fos-immunoreactive neurons that were negative for ChRGR in a region of the motor cortex. CONCLUSIONS/SIGNIFICANCE: The opto-current-clamp study suggests that the depolarization of a small number of neurons

Highlights: • 14,15-EET inhibits OGD-induced apoptosis in corticalneurons. • Mitochondrial biogenesis of corticalneurons is promoted by 14,15-EET. • 14,15-EET preserves mitochondrial function of corticalneurons under OGD. • CREB mediates effect of 14,15-EET on mitochondrial biogenesis and function. - Abstract: 14,15-Epoxyeicosatrienoic acid (14,15-EET), a metabolite of arachidonic acid, is enriched in the brain cortex and exerts protective effect against neuronal apoptosis induced by ischemia/reperfusion. Although apoptosis has been well recognized to be closely associated with mitochondrial biogenesis and function, it is still unclear whether the neuroprotective effect of 14,15-EET is mediated by promotion of mitochondrial biogenesis and function in corticalneurons under the condition of oxygen–glucose deprivation (OGD). In this study, we found that 14,15-EET improved cell viability and inhibited apoptosis of corticalneurons. 14,15-EET significantly increased the mitochondrial mass and the ratio of mitochondrial DNA to nuclear DNA. Key makers of mitochondrial biogenesis, peroxisome proliferator activator receptor gamma-coactivator 1 alpha (PGC-1α), nuclear respiratory factor 1 (NRF-1) and mitochondrial transcription factor A (TFAM), were elevated at both mRNA and protein levels in the corticalneurons treated with 14,15-EET. Moreover, 14,15-EET markedly attenuated the decline of mitochondrial membrane potential, reduced ROS, while increased ATP synthesis. Knockdown of cAMP-response element binding protein (CREB) by siRNA blunted the up-regulation of PGC-1α and NRF-1 stimulated by 14,15-EET, and consequently abolished the neuroprotective effect of 14,15-EET. Our results indicate that 14,15-EET protects neurons from OGD-induced apoptosis by promoting mitochondrial biogenesis and function through CREB mediated activation of PGC-1α and NRF-1.

Seizure activity is linked to astrocyte activation as well as dysfunctional corticalneuron excitability produced from changes in calcium-activated potassium (KCa) channel function. Ciliary neurotrophic factor-treated astrocyte conditioned medium (CNTF-ACM) can be used to investigate the peripheral effects of activated astrocytes upon corticalneurons. However, CNTF-ACM's effect upon KCa channel activity in cultured corticalneurons has not yet been investigated. Whole-cell patch clamp recordings were performed in rat corticalneurons to evaluate CNTF-ACM's effects upon charybdotoxin-sensitive large-conductance KCa (BK) channel currents and apamin-sensitive small-conductance KCa (SK) channel current. Biotinylation and RT-PCR were applied to assess CNTF-ACM's effects upon the protein and mRNA expression, respectively, of the SK channel subunits SK2 and SK3 and the BK channel subunits BKα1 and BKβ3. An anti-fibroblast growth factor-2 (FGF-2) monoclonal neutralizing antibody was used to assess the effects of the FGF-2 component of CNTF-ACM. CNTF-ACM significantly increased KCa channel current density, which was predominantly attributable to gains in BK channel activity (p ACM produced a significant increase in BKα1 and BKβ3 expression (p 0.05). Blocking FGF-2 produced significant reductions in KCa channel current density (p > 0.05) as well as BKα1 and BKβ3 expression in CNTF-ACM-treated neurons (p > 0.05). CNTF-ACM significantly enhances BK channel activity in rat corticalneurons and that FGF-2 is partially responsible for these effects. CNTF-induced astrocyte activation results in secretion of neuroactive factors which may affect neuronal excitability and resultant seizure activity in mammalian corticalneurons.

In this paper we provide, for the first time to our knowledge, the effective orientation of the SHG source in cultured corticalneuronal processes in vitro. This is done by the use of the polarization sensitive second harmonic generation (PSHG) imaging microscopy technique. By performing a pixel-level resolution analysis we found that the SHG dipole source has a distribution of angles centered at θe =33.96°, with a bandwidth of ∆θe = 12.85°. This orientation can be related with the molecular...

Neurotrophic factors yield neuroprotection by mechanisms that may be related to their effects as inhibitors of apoptosis as well as their effects on ion channels. The effect of ciliary neurotrophic factor (CNTF) on high-threshold voltage-activated Ca channels in cultured fetal mouse brain cortical...... neurones was investigated. Addition of CNTF into serum-free growth medium resulted in delayed reduction of the Ca2+ currents. The currents decreased to 50% after 4 h and stabilized at this level during incubation with CNTF for 48 h. Following removal of CNTF the inhibition was completely reversed after 18...

Full Text Available &lt;b&gt;Purpose:&lt;/b&gt; Hypoxic-ischemic encephalopathy is an important cause of neonatal mortality, as this brain injury disrupts normal mitochondrial respiratory activity. Carnitine plays an essential role in mitochondrial fatty acid transport and modulates excess acyl coenzyme A levels. In this study, we investigated whether treatment of primary cultures of rat corticalneurons with L-carnitine was able to prevent neurotoxicity resulting from oxygen-glucose deprivation (OGD. &lt;b&gt;Methods:&lt;/b&gt; Corticalneurons were prepared from Sprague-Dawley rat embryos. L-Carnitine was applied to cultures just prior to OGD and subsequent reoxygenation. The numbers of cells that stained with acridine orange (AO and propidium iodide (PI were counted, and lactate dehydrogenase (LDH activity and reactive oxygen species (ROS levels were measured. The 3-(4,5-dimethylthiazol-2-yl-2,5- diphenyltetrazolium bromide assay and the terminal uridine deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assay were performed to evaluate the effect of L-carnitine (1 μM, 10 μM, and 100 μM on OGD-induced neurotoxicity. &lt;B&gt;Results:&lt;/b&gt; Treatment of primary cultures of rat corticalneurons with L-carnitine significantly reduced cell necrosis and prevented apoptosis after OGD. L-Carnitine application significantly reduced the number of cells that died, as assessed by the PI/AO ratio, and also reduced ROS release in the OGD groups treated with 10 μM and 100 μM of L-carnitine compared with the untreated OGD group (P&lt;0.05. The application of L-carnitine at 100 μM significantly decreased cytotoxicity, LDH release, and inhibited apoptosis compared to the untreated OGD group (P&lt;0.05. &lt;B&gt;Conclusion:&lt;/b&gt; L-Carnitine has neuroprotective benefits against OGD in rat primary corticalneurons in vitro.

In migrating neurons, the centrosome nucleates and anchors a polarized network of microtubules that directs organelle movements. We report here that the mother centriole of neurons migrating tangentially from the medial ganglionic eminence (MGE) assembles a short primary cilium and exposes this cilium to the cell surface by docking to the plasma membrane in the leading process. Primary cilia are built by intraflagellar transport (IFT), which is also required for Sonic hedgehog (Shh) signal transduction in vertebrates. We show that Shh pathway perturbations influenced the leading process morphology and dynamics of MGE cells. Whereas Shh favored the exit of MGE cells away from their tangential migratory paths in the developing cortex, cyclopamine or invalidation of IFT genes maintained MGE cells in the tangential paths. Our findings show that signals transmitted through the primary cilium promote the escape of future GABAergic interneurons from their tangential routes to colonize the cortical plate.

Objective To research the effect of melatonin against glutamate excitotoxicity. Methods The model of glutamate-induced excitotoxic damage was built up in rat cerebral cortical cell culture. The effect of mela- tonin against excitotoxic injury was observed by determining the leakage rate of lactate dehydrogenase(LDH) from neurons. Results The leakage rate of LDH wasn＇t decreased markedly when cultures were exposed to melatonin be- fore, during or 6 h after glutamate treatment. The leakage rate of LDH was decreased significantly when melatonin was administered 0 h, 2 h or 4 h after the cultures were exposed to glutamate. The inhibitory function of melatonin on LDH leakage was most effective at 2 h and 4 h. Conclusion Melatonin has protective effects on neurons damaged by glutamate in a certain time limit.

The golli proteins, products of the myelin basic protein gene, are widely expressed in oligodendrocyte progenitor cells and neurons during the postnatal development of the brain. While golli appears to be important for oligodendrocyte migration and differentiation, its function in neuronal development is completely unknown. We have found that golli proteins function as new and novel modulators of voltage-operated Ca(++) channels (VOCCs) in neurons. In vitro, golli knock-out (KO) neurons exhibit decreased Ca(++) influx after plasma membrane depolarization and a substantial maturational delay. Increased expression of golli proteins enhances L-type Ca(++) entry and processes outgrowth in corticalneurons, and pharmacological activation of L-type Ca(++) channels stimulates maturation and prevents cell death in golli-KO neurons. In situ, Ca(++) influx mediated by L-type VOCCs was significantly decreased in cortical and hippocampal neurons of the golli-KO brain. These Ca(++) alterations affect cortical and hippocampal development and the proliferation and survival of neural progenitor cells during the postnatal development of the golli-KO brain. The CA1/3 sections and the dentate gyrus of the hippocampus were reduced in the golli-KO mice as well as the density of dendrites in the somatosensory cortex. Furthermore, the golli-KO mice display abnormal behavior including deficits in episodic memory and reduced anxiety. Because of the expression of the golli proteins within neurons in learning and memory centers of the brain, this work has profound implication in neurodegenerative diseases and neurological disorders.

Full Text Available Glutamatergic neurons of the mammalian cerebral cortex originate from the radial glia (RG progenitors in the ventricular zone (VZ. During corticogenesis, neuroblasts migrate toward the pial surface using two different migration modes. One is multipolar (MP migration with random directional movement, and the other is locomotion, which is a unidirectional movement guided by the RG fiber. After reaching their final destination, the neurons finalize their migration by terminal translocation, which is followed by maturation via dendrite extension to initiate synaptogenesis and thereby complete neural circuit formation. This switching of migration modes during cortical development is unique in mammals, which suggests that the RG-guided locomotion mode may contribute to the evolution of the mammalian neocortical 6-layer structure. Many factors have been reported to be involved in the regulation of this radial neuronal migration process. In general, the radial migration can be largely divided into four steps; (1 maintenance and departure from the VZ of neural progenitor cells, (2 MP migration and transition to bipolar cells, (3 RG-guided locomotion, and (4 terminal translocation and dendrite maturation. Among these, many different gene mutations or knockdown effects have resulted in failure of the MP to bipolar transition (step 2, suggesting that it is a critical step, particularly in radial migration. Moreover, this transition occurs at the subplate layer. In this review, we summarize recent advances in our understanding of the molecular mechanisms underlying each of these steps. Finally, we discuss the evolutionary aspects of neuronal migration in corticogenesis.

Full Text Available A computational model of a self-structuring neuronal net is presented in which repetitively applied pattern sets induce the formation of cortical columns and microcircuits which decode distinct patterns after a learning phase. In a case study, it is demonstrated how specific neurons in a feature classifier layer become orientation selective if they receive bar patterns of different slopes from an input layer. The input layer is mapped and intertwined by self-evolving neuronal microcircuits to the feature classifier layer. In this topical overview, several models are discussed which indicate that the net formation converges in its functionality to a mathematical transform which maps the input pattern space to a feature representing output space. The self-learning of the mathematical transform is discussed and its implications are interpreted. Model assumptions are deduced which serve as a guide to apply model derived repetitive stimuli pattern sets to in vitro cultures of neuron ensembles to condition them to learn and execute a mathematical transform.

Axonal regeneration in the central nervous system is prevented, in part, by inhibitory proteins expressed by myelin, including Myelin-associated glycoprotein (MAG). Although injury to the corticospinal tract can result in permanent disability, little is known regarding the mechanisms by which MAG affects corticalneurons. Here, we demonstrate that corticalneurons plated on MAG expressing CHO cells, exhibit a striking reduction in process outgrowth. Interestingly, none of the receptors previously implicated in MAG signaling, including the p75 neurotrophin receptor or gangliosides, contributed significantly to MAG-mediated inhibition. However, blocking the small GTPase Rho or its downstream effector kinase, ROCK, partially reversed the effects of MAG on the neurons. In addition, we identified the lipid phosphatase PTEN as a mediator of MAG’s inhibitory effects on neurite outgrowth. Knockdown or gene deletion of PTEN or over expression of activated AKT in corticalneurons resulted in significant, although partial, rescue of neurite outgrowth on MAG-CHO cells. Moreover, MAG decreased the levels of phospho-Akt, suggesting that it activates PTEN in the neurons. Taken together, these results suggest a novel pathway activated by MAG in corticalneurons involving the PTEN/PI3K/AKT axis. PMID:20869442

Intraventricular administration of the immunotoxin 192 IgG-saporin in rats has been shown to cause a selective loss of cholinergic afferents to the hippocampus and cortical areas, and to facilitate seizure development in hippocampal kindling. Here we demonstrate that this lesion also accelerates seizure progression when kindling is induced by electrical stimulations in the amygdala. However, whereas intraventricular 192 IgG-saporin facilitated the development of the initial stages of hippocampal kindling, the same lesion promoted the late stages of amygdala kindling. To explore the role of various parts of the basal forebrain cholinergic system in amygdala kindling, selective lesions of the cholinergic projections to either hippocampus or cortex were produced by intraparenchymal injections of 192 IgG-saporin into medial septum/vertical limb of the diagonal band or nucleus basalis, respectively. Cholinergic denervation of the cortical regions caused acceleration of amygdala kindling closely resembling that observed after the more widespread lesion induced by intraventricular 192 IgG-saporin. In contrast, removal of the cholinergic input to the hippocampus had no effect on the development of amygdala kindling. These data indicate that basal forebrain cholinergic neurons suppress kindling elicited from amygdala, and that this dampening effect is mediated via cortical but not hippocampal projections.

1. The muscarinic receptor antagonists gallamine and pirenzepine were iontophoretically applied to rat cerebral cortical cholinoceptive neurones, including corticospinal neurones, to assess their effects on spontaneous firing, and firing induced by: stimulation of the nucleus basalis magnocellularis (NBM); contralateral hindpaw stimulation; application of acetylcholine (ACh); and application of glutamate. 2. Both compounds potently inhibited firing induced by ACh iontophoresis, whilst neither compound consistently altered firing induced by application of glutamate. 3. Gallamine was very effective and pirenzepine less effective, at inhibiting both spontaneous firing and the delayed firing induced by NBM stimulation. The short-latency excitations elicited by NBM stimulation were enhanced by these muscarinic antagonists. 4. Gallamine and pirenzepine enhanced cortical cholinoceptive cell firing induced by contralateral hindpaw stimulation. 5. It is concluded that gallamine depresses spontaneous activity more than pirenzepine, and that both compounds can affect the cortical cell firing evoked by stimulation of the NBM and of thalamo-cortical afferent fibres. PMID:3401638

Because the excitable properties of neurons in the neocortex depend on the characteristics of voltage-gated Na(+) channels, factors which regulate those characteristics can fundamentally modify the dynamics of cortical circuits. Here, we report on a novel neuromodulatory mechanism that links the availability of Na(+) channels to metabolism of polyamines (PAs) in the cerebral cortex. Using single channel and whole-cell recordings, we found that products of PA metabolism, the ubiquitous aliphatic polycations spermine and spermidine, are endogenous blockers of Na(+) channels in layer 5 pyramidal cells. Because the blockade is activity-dependent, it is particularly effective against Na(+) channels which fail to inactivate rapidly and thus underlie the persistent Na(+) current. At the level of the local cortical circuit, pharmacological depletion of PAs led to increased spontaneous spiking and periods of hypersynchronous discharge. Our data suggest that changes in PA levels, whether associated with normal brain states or pathological conditions, profoundly modify Na(+) channel availability and thereby shape the integrative behavior of single neurons and neocortical circuits.

Lesioned axons do not regenerate in the adult mammalian CNS, owing to the over-expression of inhibitory molecules such as myelin-derived proteins or chondroitin sulphate proteoglycans. In order to overcome axon inhibition, strategies based on extrinsic and intrinsic treatments have been developed. For myelin-associated inhibition, blockage with NEP1-40, receptor bodies or IN-1 antibodies has been used. In addition, endogenous blockage of cell signalling mechanisms induced by myelin-associated proteins is a potential tool for overcoming axon inhibitory signals. We examined the participation of glycogen synthase kinase 3beta (GSK3beta) and extracellular-related kinase (ERK) 1/2 in axon regeneration failure in lesioned corticalneurons. We also investigated whether pharmacological blockage of GSK3beta and ERK1/2 activities facilitates regeneration after myelin-directed inhibition in two models: (i) cerebellar granule cells and (ii) lesioned entorhino-hippocampal pathway in slice cultures, and whether the regenerative effects are mediated by Nogo Receptor 1 (NgR1). We demonstrate that, in contrast to ERK1/2 inhibition, the pharmacological treatment of GSK3beta inhibition strongly facilitated regrowth of cerebellar granule neurons over myelin independently of NgR1. Finally, these regenerative effects were corroborated in the lesioned entorhino-hippocampal pathway in NgR1-/- mutant mice. These results provide new findings for the development of new assays and strategies to enhance axon regeneration in injured cortical connections.

Recent studies have revealed that in vivo corticalneurons show spontaneous transitions between two subthreshold levels of the membrane potentials, 'up' and 'down' states. The neural mechanism of generating those spontaneous states transitions, however, remains unclear. Recent electrophysiological studies have suggested that those state transitions may occur through activation of a hyperpolarization-activated cation current (H-current), possibly by inhibitory synaptic inputs. Here, we demonstrate that two-state membrane potential fluctuations similar to those exhibited by in vivo neurons can be generated through a spike-timing-dependent self-organizing process in a network of inhibitory neurons and excitatory neurons expressing the H-current.

Estrogens are important regulators of bone mass and their effects are mainly mediated via estrogen receptor (ER)α. Central ERα exerts an inhibitory role on bone mass. ERα is highly expressed in the arcuate (ARC) and the ventromedial (VMN) nuclei in the hypothalamus. To test whether ERα in proopiomelanocortin (POMC) neurons, located in ARC, is involved in the regulation of bone mass, we used mice lacking ERα expression specifically in POMC neurons (POMC-ERα(-/-)). Female POMC-ERα(-/-) and control mice were ovariectomized (OVX) and treated with vehicle or estradiol (0.5 μg/d) for 6 weeks. As expected, estradiol treatment increased the cortical bone thickness in femur, the cortical bone mechanical strength in tibia and the trabecular bone volume fraction in both femur and vertebrae in OVX control mice. Importantly, the estrogenic responses were substantially increased in OVX POMC-ERα(-/-) mice compared with the estrogenic responses in OVX control mice for cortical bone thickness (+126 ± 34%, P mass, ERα was silenced using an adeno-associated viral vector. Silencing of ERα in hypothalamic VMN resulted in unchanged bone mass. In conclusion, mice lacking ERα in POMC neurons display enhanced estrogenic response on cortical bone mass and mechanical strength. We propose that the balance between inhibitory effects of central ERα activity in hypothalamic POMC neurons in ARC and stimulatory peripheral ERα-mediated effects in bone determines cortical bone mass in female mice.

Full Text Available The cortex is a complex system, characterized by its dynamics and architecture, which underlie many functions such as action, perception, learning, language, and cognition. Its structural architecture has been studied for more than a hundred years; however, its dynamics have been addressed much less thoroughly. In this paper, we review and integrate, in a unifying framework, a variety of computational approaches that have been used to characterize the dynamics of the cortex, as evidenced at different levels of measurement. Computational models at different space-time scales help us understand the fundamental mechanisms that underpin neural processes and relate these processes to neuroscience data. Modeling at the single neuron level is necessary because this is the level at which information is exchanged between the computing elements of the brain; the neurons. Mesoscopic models tell us how neural elements interact to yield emergent behavior at the level of microcolumns and cortical columns. Macroscopic models can inform us about whole brain dynamics and interactions between large-scale neural systems such as cortical regions, the thalamus, and brain stem. Each level of description relates uniquely to neuroscience data, from single-unit recordings, through local field potentials to functional magnetic resonance imaging (fMRI, electroencephalogram (EEG, and magnetoencephalogram (MEG. Models of the cortex can establish which types of large-scale neuronal networks can perform computations and characterize their emergent properties. Mean-field and related formulations of dynamics also play an essential and complementary role as forward models that can be inverted given empirical data. This makes dynamic models critical in integrating theory and experiments. We argue that elaborating principled and informed models is a prerequisite for grounding empirical neuroscience in a cogent theoretical framework, commensurate with the achievements in the

The release of the serine proteinase tissue-type plasminogen activator (tPA) from the presynaptic terminal of cerebral corticalneurons plays a central role in the development of synaptic plasticity, adaptation to metabolic stress and neuronal survival. Our earlier studies indicate that by inducing the recruitment of the cytoskeletal protein βII-spectrin and voltage-gated calcium channels to the active zone, tPA promotes Ca(2+)-dependent translocation of synaptic vesicles (SVs) to the synaptic release site where they release their load of neurotransmitters into the synaptic cleft. Here we used a combination of in vivo and in vitro experiments to investigate whether this effect leads to depletion of SVs in the presynaptic terminal. Our data indicate that tPA promotes SV endocytosis via a mechanism that does not require the conversion of plasminogen into plasmin. Instead, we show that tPA induces calcineurin-mediated dynamin I dephosphorylation, which is followed by dynamin I-induced recruitment of the actin-binding protein profilin II to the presynaptic membrane, and profilin II-induced F-actin formation. We report that this tPA-induced sequence of events leads to the association of newly formed SVs with F-actin clusters in the endocytic zone. In summary, the data presented here indicate that following the exocytotic release of neurotransmitters tPA activates the mechanism whereby SVs are retrieved from the presynaptic membrane and endocytosed to replenish the pool of vesicles available for a new cycle of exocytosis. Together, these results indicate that in murine cerebral corticalneurons tPA plays a central role coupling SVs exocytosis and endocytosis.

Considerable progress has been made in converting human pluripotent stem cells (hPSCs) into functional neurons. However, the protracted timing of human neuron specification and functional maturation remains a key challenge that hampers the routine application of hPSC-derived lineages in disease modeling and regenerative medicine. Using a combinatorial small-molecule screen, we previously identified conditions to rapidly differentiate hPSCs into peripheral sensory neurons. Here we generalize the approach to central nervous system (CNS) fates by developing a small-molecule approach for accelerated induction of early-born corticalneurons. Combinatorial application of six pathway inhibitors induces post-mitotic corticalneurons with functional electrophysiological properties by day 16 of differentiation, in the absence of glial cell co-culture. The resulting neurons, transplanted at 8 d of differentiation into the postnatal mouse cortex, are functional and establish long-distance projections, as shown using iDISCO whole-brain imaging. Accelerated differentiation into corticalneuron fates should facilitate hPSC-based strategies for disease modeling and cell therapy in CNS disorders.

BACKGROUND:Numerous current studies have suggested that human telomerase reverse transcriptase (hTERT) gene has neuroprotective effects and can inhibit apoptosis induced by various cytotoxic stresses;however,the mechanism of action remains unknown.OBJECTIVE:To evaluate the neuroprotective effects and possible mechanism of action of hTERT gene transfection in human embryonic corticalneurons treated with beta-amyloid fragment 25-35 (Aβ25-35).DESIGN,TIME AND SETTING:The randomized,controlled and molecular biological studies were performed at the Department of Anatomy and Brain Research,Zhongshan School of Medicine,Sun Yat-sen University,China,from September 2005 to June 2008.MATERIALS:AdEasy-1 Expression System was gifted by Professor Guoquan Gao from Sun Yat-Sen University,China.Human corticalneurons were derived from 12-20 week old aborted fetuses,obtained from the Guangzhou Maternal and Child Health Hospital,China.Mouse anti-Cdk5 and mouse anti-p16 monoclonal antibodies (Lab Vision,USA),and mouse anti-hTERT monoclonal antibody (Epitomics,USA),were used in this study.METHODS:(1) Recombinant adenovirus vectors,encoding hTERT (Ad-hTERT) and green fluorescent protein (Ad-GFP),were constructed using the AdEasy-1 Expression System.Human embryonic corticalneurons in the Ad-hTERT group were transfected with Ad-hTERT for 1-21 days.Likewise,human embryonic corticalneurons in the Ad-GFP group were transfected with Ad-GFP for 1-21 days.Human embryonic corticalneurons in the control group were cultured as normal.(2) Human embryonic corticalneurons in the Ad-hTERT group were treated with 10 μmol/L Aβ25-35 for 24 hours.Normal human embryonic corticalneurons treated with 10 μmol/L Aβ25-35 for 24 hours served as a model group.Human embryonic corticalneurons in the Ad-GFP and control groups were not treated with Aβ25-35.MAIN OUTCOME MEASURES:Expression of hTERT in human embryonic corticalneurons was evaluated by immunocytochemical staining and Western blot assay

Full Text Available Curcumin is a molecule found in turmeric root that has anti-inflammatory, antioxidant, and anti-tumor properties and has been widely used as both an herbal drug and a food additive to treat or prevent neurodegenerative diseases. To explore whether curcumin is able to ameliorate HIV-1-associated neurotoxicity, we treated a murine microglial cell line (N9 and primary rat corticalneurons with curcumin in the presence or absence of neurotoxic HIV-1 gp120 (V3 loop protein. We found that HIV-1 gp120 profoundly induced N9 cells to produce reactive oxygen species (ROS, tumor necrosis factor-α (TNF-α and monocyte chemoattractant protein-1 (MCP-1. HIV-1 gp120 also induced apoptosis of primary rat corticalneurons. Curcumin exerted a powerful inhibitory effect against HIV-1 gp120-induced neuronal damage, reducing the production of ROS, TNF-α and MCP-1 by N9 cells and inhibiting apoptosis of primary rat corticalneurons. Curcumin may exert its biological activities through inhibition of the delayed rectification and transient outward potassium (K(+ current, as curcumin effectively reduced HIV-1 gp120-mediated elevation of the delayed rectification and transient outward K(+ channel current in neurons. We conclude that HIV-1 gp120 increases ROS, TNF-α and MCP-1 production in microglia, and induces corticalneuron apoptosis by affecting the delayed rectification and transient outward K(+ channel current. Curcumin reduces production of ROS and inflammatory mediators in HIV-1-gp120-stimulated microglia, and protects corticalneurons against HIV-1-mediated apoptosis, most likely through inhibition of HIV-1 gp120-induced elevation of the delayed rectification and transient outward K(+ current.

1. The fluctuations during various phases of the slow sleep oscillation (< 1 Hz) in synaptic responsiveness of motor cortical (Cx), thalamic reticular (RE) and thalamocortical (TC) neurones were investigated intracellularly in cats under ketamine-xylazine anaesthesia. Orthodromic responses to stimuli applied to brachium conjunctivum (BC) axons and corticothalamic pathways were studied. The phases of slow oscillation consist of a long-hyperpolarized, followed by a sharp depth-negative EEG deflection and a series of faster waves that are associated with the depolarization of Cx and RE neurones, while TC cells display a sequence of IPSPs within the spindle frequency. 2. BC-evoked bisynaptic excitatory postsynaptic potentials (EPSPs) in Cx and RE neurones were drastically reduced in amplitude during the long-lasting hyperpolarization and the early part of the depolarizing phase. By contrast, the BC-evoked monosynaptic EPSPs of TC cells were not diminished during the depth-positive EEG wave, but the hyperpolarization during this phase of the slow oscillation prevented TC neurones transferring prethalamic signals to the cortex. 3. At variance with the diminished bisynaptic EPSPs evoked in response to BC stimuli during the long-lasting hyperpolarization, Cx-evoked monosynaptic EPSPs in Cx cells increased linearly with hyperpolarization during this phase of the slow oscillation. Similarly, the amplitudes of Cx-evoked EPSPs in RE and TC cells were not diminished during the long-lasting hyperpolarization. 4. The diminished responsiveness of Cx and RE neurones to prethalamic volleys during the long-lasting hyperpolarization is attributed to gating processes at the level of TC cells that, because of their hyperpolarization, do not transfer prethalamic information to further relays. PMID:8814620

Neural interfaces aim to restore neurological function lost during disease or injury. Novel implantable neural interfaces increasingly capitalize on novel materials to achieve microscale coupling with the nervous system. Like any biomedical device, neural interfaces should consist of materials that exhibit biocompatibility in accordance with the international standard ISO10993-5, which describes in vitro testing involving fibroblasts where cytotoxicity serves as the main endpoint. In the present study, we examine the utility of living neuronal networks as functional assays for in vitro material biocompatibility, particularly for materials that comprise implantable neural interfaces. Embryonic mouse cortical tissue was cultured to form functional networks where spontaneous action potentials, or spikes, can be monitored non-invasively using a substrate-integrated microelectrode array. Taking advantage of such a platform, we exposed established positive and negative control materials to the neuronal networks in a consistent method with ISO 10993-5 guidance. Exposure to the negative controls, gold and polyethylene, did not significantly change the neuronal activity whereas the positive controls, copper and polyvinyl chloride (PVC), resulted in reduction of network spike rate. We also compared the functional assay with an established cytotoxicity measure using L929 fibroblast cells. Our findings indicate that neuronal networks exhibit enhanced sensitivity to positive control materials. In addition, we assessed functional neurotoxicity of tungsten, a common microelectrode material, and two conducting polymer formulations that have been used to modify microelectrode properties for in vivo recording and stimulation. These data suggest that cultured neuronal networks are a useful platform for evaluating the functional toxicity of materials intended for implantation in the nervous system.

Aluminum (Al) exposure and apoptotic cell death have been implicated in several neurodegenerative diseases.the mechanisms by which Al interacts with the nervous system are only partly understood.In this study,we used cultured corticalneurons to investigate the ability of Al to induce the apoptosis of neurons and to explore the role of SAPK/JNK signal transduction pathway on the apoptosis induced by Al.It was found that Al-induced degeneration of corticalneurons involved the DNA fragmentation characteristic of apoptosis.The rate of apoptosis increased significantly,which was measured by TdT-mediated dUTKP nick end labeling.Westerm blot analysis showed that SAPK/JNK activities of corticalneurons varied when the dose and exposure time of AlCl3 were different.Our study demonstrates that Al can induce the apoptosis of corticalneurons and SAPK/JNK signal transduction pathway may play a great role in the apoptosis.

Radiofrequency electromagnetic fields (EMF) are harmful to public health, but the certain anti-irradiation mechanism is not clear yet. The present study was performed to investigate the possible protective effects of green tea polyphenols against electromagnetic radiation-induced injury in the cultured rat corticalneurons. In this study, green tea polyphenols were used in the cultured corticalneurons exposed to 1800 MHz EMFs by the mobile phone. We found that the mobile phone irradiation for 24 h induced marked neuronal cell death in the MTT (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl-tetrazolium bromide) and TUNEL (TdT mediated biotin-dUTP nicked-end labeling) assay, and protective effects of green tea polyphenols on the injured corticalneurons were demonstrated by testing the content of Bcl-2 Assaciated X protein (Bax) in the immunoprecipitation assay and Western blot assay. In our study results, the mobile phone irradiation-induced increases in the content of active Bax were inhibited significantly by green tea polyphenols, while the contents of total Bax had no marked changes after the treatment of green tea polyphenols. Our results suggested a neuroprotective effect of green tea polyphenols against the mobile phone irradiation-induced injury on the cultured rat corticalneurons.

Full Text Available Abstract Background Exposure to ethanol during early development triggers severe neuronal death by activating multiple stress pathways and causes neurological disorders, such as fetal alcohol effects or fetal alcohol syndrome. This study investigated the effect of ethanol on intracellular events that predispose developing neurons for apoptosis via calcium-mediated signaling. Although the underlying molecular mechanisms of ethanol neurotoxicity are not completely determined, mitochondrial dysfunction, altered calcium homeostasis and apoptosis-related proteins have been implicated in ethanol neurotoxicity. The present study was designed to evaluate the neuroprotective mechanisms of metformin (Met and thymoquinone (TQ during ethanol toxicity in rat prenatal corticalneurons at gestational day (GD 17.5. Results We found that Met and TQ, separately and synergistically, increased cell viability after ethanol (100 mM exposure for 12 hours and attenuated the elevation of cytosolic free calcium [Ca2+]c. Furthermore, Met and TQ maintained normal physiological mitochondrial transmembrane potential (ΔψM, which is typically lowered by ethanol exposure. Increased cytosolic free [Ca2+]c and lowered mitochondrial transmembrane potential after ethanol exposure significantly decreased the expression of a key anti-apoptotic protein (Bcl-2, increased expression of Bax, and stimulated the release of cytochrome-c from mitochondria. Met and TQ treatment inhibited the apoptotic cascade by increasing Bcl-2 expression. These compounds also repressed the activation of caspase-9 and caspase-3 and reduced the cleavage of PARP-1. Morphological conformation of cell death was assessed by TUNEL, Fluoro-Jade-B, and PI staining. These staining methods demonstrated more cell death after ethanol treatment, while Met, TQ or Met plus TQ prevented ethanol-induced apoptotic cell death. Conclusion These findings suggested that Met and TQ are strong protective agents against ethanol

Alterations in the cellular metabolic machinery of the brain are associated with neurodegenerative disorders such as Alzheimer's disease. Novel human cellular disease models are essential in order to study underlying disease mechanisms. In the present study, we characterized major metabolic pathways in neurons derived from human induced pluripotent stem cells (hiPSC). With this aim, cultures of hiPSC-derived neurons were incubated with [U-(13)C]glucose, [U-(13)C]glutamate or [U-(13)C]glutamine. Isotopic labeling in metabolites was determined using gas chromatography coupled to mass spectrometry, and cellular amino acid content was quantified by high-performance liquid chromatography. Additionally, we evaluated mitochondrial function using real-time assessment of oxygen consumption via the Seahorse XF(e)96 Analyzer. Moreover, in order to validate the hiPSC-derived neurons as a model system, a metabolic profiling was performed in parallel in primary neuronal cultures of mouse cerebral cortex and cerebellum. These serve as well-established models of GABAergic and glutamatergic neurons, respectively. The hiPSC-derived neurons were previously characterized as being forebrain-specific cortical glutamatergic neurons. However, a comparable preparation of predominantly mouse cortical glutamatergic neurons is not available. We found a higher glycolytic capacity in hiPSC-derived neurons compared to mouse neurons and a substantial oxidative metabolism through the mitochondrial tricarboxylic acid (TCA) cycle. This finding is supported by the extracellular acidification and oxygen consumption rates measured in the cultured human neurons. [U-(13)C]Glutamate and [U-(13)C]glutamine were found to be efficient energy substrates for the neuronal cultures originating from both mice and humans. Interestingly, isotopic labeling in metabolites from [U-(13)C]glutamate was higher than that from [U-(13)C]glutamine. Although the metabolic profile of hiPSC-derived neurons in vitro was

Electrical brain stimulation (EBS) is an emerging therapy for the treatment of neurological disorders, and computational modeling studies of EBS have been used to determine the optimal parameters for highly cost-effective electrotherapy. Recent notable growth in computing capability has enabled researchers to consider an anatomically realistic head model that represents the full head and complex geometry of the brain rather than the previous simplified partial head model (extruded slab) that represents only the precentral gyrus. In this work, subdural cortical stimulation (SuCS) was found to offer a better understanding of the differential activation of corticalneurons in the anatomically realistic full-head model than in the simplified partial-head models. We observed that layer 3 pyramidal neurons had comparable stimulation thresholds in both head models, while layer 5 pyramidal neurons showed a notable discrepancy between the models; in particular, layer 5 pyramidal neurons demonstrated asymmetry in the thresholds and action potential initiation sites in the anatomically realistic full-head model. Overall, the anatomically realistic full-head model may offer a better understanding of layer 5 pyramidal neuronal responses. Accordingly, the effects of using the realistic full-head model in SuCS are compelling in computational modeling studies, even though this modeling requires substantially more effort.

Modifying the density and distribution of ion channels in a neuron (by natural up- and down-regulation, by pharmacological intervention or by spontaneous mutations) changes its activity pattern. In the present investigation, we analyze how the impulse patterns are regulated by the density of voltage-gated channels in a model neuron, based on voltage clamp measurements of hippocampal interneurons. At least three distinct oscillatory patterns, associated with three distinct regions in the Na-K channel density plane, were found. A stability analysis showed that the different regions are characterized by saddle-node, double-orbit, and Hopf bifurcation threshold dynamics, respectively. Single strongly graded action potentials occur in an area outside the oscillatory regions, but less graded action potentials occur together with repetitive firing over a considerable range of channel densities. The presently found relationship between channel densities and oscillatory behavior may be relevance for understanding principal spiking patterns of corticalneurons (regular firing and fast spiking). It may also be of relevance for understanding the action of pharmacological compounds on brain oscillatory activity.

Full Text Available The ability to discriminate tones of different frequencies is fundamentally important for everyday hearing. While neurons in the primary auditory cortex (AC respond differentially to tones of different frequencies, whether and how AC regulates auditory behaviors that rely on frequency discrimination remains poorly understood. Here, we find that the level of activity of inhibitory neurons in AC controls frequency specificity in innate and learned auditory behaviors that rely on frequency discrimination. Photoactivation of parvalbumin-positive interneurons (PVs improved the ability of the mouse to detect a shift in tone frequency, whereas photosuppression of PVs impaired the performance. Furthermore, photosuppression of PVs during discriminative auditory fear conditioning increased generalization of conditioned response across tone frequencies, whereas PV photoactivation preserved normal specificity of learning. The observed changes in behavioral performance were correlated with bidirectional changes in the magnitude of tone-evoked responses, consistent with predictions of a model of a coupled excitatory-inhibitory cortical network. Direct photoactivation of excitatory neurons, which did not change tone-evoked response magnitude, did not affect behavioral performance in either task. Our results identify a new function for inhibition in the auditory cortex, demonstrating that it can improve or impair acuity of innate and learned auditory behaviors that rely on frequency discrimination.

Brains affected by Alzheimer's disease (AD) show a large spectrum of mitochondrial alterations at both morphological and genetic level. The causal link between β-amyloid (Aβ) and mitochondrial dysfunction has been established in cellular models of AD. We observed previously that lycopene, a member of the carotenoid family of phytochemicals, could counteract neuronal apoptosis and cell damage induced by Aβ and other neurotoxic substances, and that this neuroprotective action somehow involved the mitochondria. The present study aims to investigate the effects of lycopene on mitochondria in cultured rat corticalneurons exposed to Aβ. It was found that lycopene attenuated Aβ-induced oxidative stress, as evidenced by the decreased intracellular reactive oxygen species generation and mitochondria-derived superoxide production. Additionally, lycopene ameliorated Aβ-induced mitochondrial morphological alteration, opening of the mitochondrial permeability transition pores and the consequent cytochrome c release. Lycopene also improved mitochondrial complex activities and restored ATP levels in Aβ-treated neuron. Furthermore, lycopene prevented mitochondrial DNA damages and improved the protein level of mitochondrial transcription factor A in mitochondria. Those results indicate that lycopene protects mitochondria against Aβ-induced damages, at least in part by inhibiting mitochondrial oxidative stress and improving mitochondrial function. These beneficial effects of lycopene may account for its protection against Aβ-induced neurotoxicity.

Full Text Available Blind source separation is the computation underlying the cocktail party effect--a partygoer can distinguish a particular talker's voice from the ambient noise. Early studies indicated that the brain might use blind source separation as a signal processing strategy for sensory perception and numerous mathematical models have been proposed; however, it remains unclear how the neural networks extract particular sources from a complex mixture of inputs. We discovered that neurons in cultures of dissociated rat cortical cells could learn to represent particular sources while filtering out other signals. Specifically, the distinct classes of neurons in the culture learned to respond to the distinct sources after repeating training stimulation. Moreover, the neural network structures changed to reduce free energy, as predicted by the free-energy principle, a candidate unified theory of learning and memory, and by Jaynes' principle of maximum entropy. This implicit learning can only be explained by some form of Hebbian plasticity. These results are the first in vitro (as opposed to in silico demonstration of neural networks performing blind source separation, and the first formal demonstration of neuronal self-organization under the free energy principle.

Green tea polyphenols are a natural product which has antioxidative and antiapoptotic effects. It has been shown that glutamate excitotoxicity induced oxidative stress is linked to neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. In this study we explored the neuroprotective effect of green teen polyphenols against glutamate excitotoxicity in the primary cultured corticalneurons. We found that green tea polyphenols protected against glutamate induced neurotoxicity in the corticalneurons as measured by MTT and TUNEL assays. Green tea polyphenols were then showed to inhibit the glutamate induced ROS release and SOD activity reduction in the neurons. Furthermore, our results demonstrated that green tea polyphenols restored the dysfunction of mitochondrial pro- or antiapoptotic proteins Bax, Bcl-2, and caspase-3 caused by glutamate. Interestingly, the neuroprotective effect of green tea polyphenols was abrogated when the neurons were incubated with siBcl-2. Taken together, these results demonstrated that green tea polyphenols protected against glutamate excitotoxicity through antioxidative and antiapoptotic pathways.

Stem cell-based approaches to restore function after stroke through replacement of dead neurons require the generation of specific neuronal subtypes. Loss of neurons in the cerebral cortex is a major cause of stroke-induced neurological deficits in adult humans. Reprogramming of adult human somatic cells to induced pluripotent stem cells is a novel approach to produce patient-specific cells for autologous transplantation. Whether such cells can be converted to functional corticalneurons that survive and give rise to behavioural recovery after transplantation in the stroke-injured cerebral cortex is not known. We have generated progenitors in vitro, expressing specific cortical markers and giving rise to functional neurons, from long-term self-renewing neuroepithelial-like stem cells, produced from adult human fibroblast-derived induced pluripotent stem cells. At 2 months after transplantation into the stroke-damaged rat cortex, the cortically fated cells showed less proliferation and more efficient conversion to mature neurons with morphological and immunohistochemical characteristics of a cortical phenotype and higher axonal projection density as compared with non-fated cells. Pyramidal morphology and localization of the cells expressing the cortex-specific marker TBR1 in a certain layered pattern provided further evidence supporting the cortical phenotype of the fated, grafted cells, and electrophysiological recordings demonstrated their functionality. Both fated and non-fated cell-transplanted groups showed bilateral recovery of the impaired function in the stepping test compared with vehicle-injected animals. The behavioural improvement at this early time point was most likely not due to neuronal replacement and reconstruction of circuitry. At 5 months after stroke in immunocompromised rats, there was no tumour formation and the grafted cells exhibited electrophysiological properties of mature neurons with evidence of integration in host circuitry. Our

Full Text Available Quantitative electroencephalography (qEEG showed that Alzheimer’s disease (AD is characterized by increased theta power, decreased alpha and beta power, and decreased coherence in the alpha and theta band in posterior regions. These abnormalities are thought to be associated with functional disconnections among cortical areas, death of corticalneurons, axonal pathology, and cholinergic deficits. Since transcranial Direct Current Stimulation (tDCS over the temporo-parietal area is thought to have beneficial effects in patients with AD, in this study we aimed to investigate whether tDCS benefits are related to tDCS-induced changes in cortical activity, as represented by qEEG.A weak anodal current (1.5 mA, 15 min was delivered bilaterally over the temporal-parietal lobe to 7 subjects with probable AD (Mini-Mental State Examination, MMSE score >20. EEG (21 electrodes, 10-20 international system was recorded for 5 minutes with eyes closed before (baseline, t0 and 30 minutes after anodal and cathodal tDCS ended (t1. At the same time points, patients performed a Word Recognition Task (WRT to assess working memory functions. The spectral power and the inter- and intra-hemispheric EEG coherence in different frequency bands (e.g., low frequencies, including delta and theta; high frequencies, including alpha and beta were calculated for each subject at t0 and t1. tDCS-induced changes in EEG neurophysiological markers were correlated with the performance of patients at the WRT.At baseline, qEEG features in AD patients confirmed that the decreased high frequency power was correlated with lower MMSE. After anodal tDCS, we observed an increase in the high-frequency power in the temporo-parietal area and an increase in the temporo-parieto-occipital coherence that correlated with the improvement at the WRT. In addition, cathodal tDCS produced a non-specific effect of decreased theta power all over the scalp that was not correlated with the clinical

Quantitative electroencephalography (qEEG) showed that Alzheimer's disease (AD) is characterized by increased theta power, decreased alpha and beta power, and decreased coherence in the alpha and theta band in posterior regions. These abnormalities are thought to be associated with functional disconnections among cortical areas, death of corticalneurons, axonal pathology, and cholinergic deficits. Since transcranial Direct Current Stimulation (tDCS) over the temporo-parietal area is thought to have beneficial effects in patients with AD, in this study we aimed to investigate whether tDCS benefits are related to tDCS-induced changes in cortical activity, as represented by qEEG. A weak anodal current (1.5 mA, 15 min) was delivered bilaterally over the temporal-parietal lobe to seven subjects with probable AD (Mini-Mental State Examination, MMSE score >20). EEG (21 electrodes, 10-20 international system) was recorded for 5 min with eyes closed before (baseline, t0) and 30 min after anodal and cathodal tDCS ended (t1). At the same time points, patients performed a Word Recognition Task (WRT) to assess working memory functions. The spectral power and the inter- and intra-hemispheric EEG coherence in different frequency bands (e.g., low frequencies, including delta and theta; high frequencies, including alpha and beta) were calculated for each subject at t0 and t1. tDCS-induced changes in EEG neurophysiological markers were correlated with the performance of patients at the WRT. At baseline, qEEG features in AD patients confirmed that the decreased high frequency power was correlated with lower MMSE. After anodal tDCS, we observed an increase in the high-frequency power in the temporo-parietal area and an increase in the temporo-parieto-occipital coherence that correlated with the improvement at the WRT. In addition, cathodal tDCS produced a non-specific effect of decreased theta power all over the scalp that was not correlated with the clinical observation at the WRT

Aim:To understand the mechanism of the transactivation of the epidermal growth factor receptor (EGFR) mediated by the adenosine A1 receptor (A1R).Methods:Primary cultured rat corticalneurons subjected to oxygen-glucose deprivation (OGD) and HEK293/A1R cells were treated with the A1R-specific agonist N6-cyclopentyladenosine (CPA).Phospho-EGFR,Akt,and ERK1/2 were observed by Western blot.An interaction between EGFR and AIR was detected using immunoprecipitation and immunocytochemistry.Results:The A1R agonist CPA causes protein kinase B (Akt) activation and protects primary corticalneurons from oxygen-glucose deprivation (OGD) insult.A1R and EGFR co-localize in the membranes of neurons and form an immunocomplex.A1R stimulation induces significant EGFR phosphorylation via a P13K and Src kinase signaling pathway;this stimulation provides a neuroprotective effect in corticalneurons.CPA leads to sustained phosphorylation of extracellularly regulated kinases 1 and 2 (ERK1/2) in corticalneurons,but only to transient phosphorylation in HEK 293/A1R cells.The response to the AtR agonist is mediated primarily through EGFR trans-activation that is dependent on pertussis toxin (PTX)-sensitive G1 protein and metalloproteases in HEK 293/A1R.Conclusion:A1R-mediated EGFR transactivation confers a neuroprotective effect in primary corticalneurons.P13 kinase and Src kinase play pivotal roles in this response.

During neocortical development, neurons undergo polarization, oriented migration, and layer-type-specific differentiation. The transcriptional programs underlying these processes are not completely understood. Here, we show that the transcription factor Bcl11a regulates polarity and migration of upper layer neurons. Bcl11a-deficient late-born neurons fail to correctly switch from multipolar to bipolar morphology, resulting in impaired radial migration. We show that the expression of Sema3c is increased in migrating Bcl11a-deficient neurons and that Bcl11a is a direct negative regulator of Sema3c transcription. In vivo gain-of-function and rescue experiments demonstrate that Sema3c is a major downstream effector of Bcl11a required for the cell polarity switch and for the migration of upper layer neurons. Our data uncover a novel Bcl11a/Sema3c-dependent regulatory pathway used by migrating corticalneurons.

The auditory system is capable of robust recognition of sounds in the presence of competing maskers (e.g., other voices or background music). This capability arises despite the fact that masking stimuli can disrupt neural responses at the cortical level. Since the origins of such interference effects remain unknown, in this study, we work to identify and quantify neural interference effects that originate due to masking occurring within and outside receptive fields of neurons. We record from single and multi-unit auditory sites from field L, the auditory cortex homologue in zebra finches. We use a novel method called spike timing-based stimulus filtering that uses the measured response of each neuron to create an individualized stimulus set. In contrast to previous adaptive experimental approaches, which have typically focused on the average firing rate, this method uses the complete pattern of neural responses, including spike timing information, in the calculation of the receptive field. When we generate and present novel stimuli for each neuron that mask the regions within the receptive field, we find that the time-varying information in the neural responses is disrupted, degrading neural discrimination performance and decreasing spike timing reliability and sparseness. We also find that, while removing stimulus energy from frequency regions outside the receptive field does not significantly affect neural responses for many sites, adding a masker in these frequency regions can nonetheless have a significant impact on neural responses and discriminability without a significant change in the average firing rate. These findings suggest that maskers can interfere with neural responses by disrupting stimulus timing information with power either within or outside the receptive fields of neurons.

The primary objective of this work was to assess the intrinsic nonbicarbonate buffer capacity (beta i) of cultured neurons and astrocytes and to compare the beta i values obtained to those of neocortical tissue. A second objective was to determine the pH dependence of beta i. Titration of homogenates of whole-brain cortical tissue and cultured neurons with NaOH and HCl gave beta i values of 25-30 mmol.l-1 x pH-1. The buffer capacity was essentially constant in the pH range of 6-7. Astrocytes showed a higher buffer capacity and a clear relationship between beta i and pH. However, beta i decreased when pH was reduced from 7 to 6. The beta i values derived from microspectrofluorometric studies on neurons and astrocytes were surprisingly variable, ranging from 10 to 50 mmol.l-1 x pH-1. The ammonia "step method" suggested that beta i increased dramatically when pH was lowered from 7 to 6 but the propionic "step method" failed to reveal such a pH dependence. Some techniques obviously give erroneous values for beta i, presumably because changes in buffer base concentration (due to transmembrane fluxes of H+, HCO3-, NH4+ or anions of weak acids) violate the principles upon which the calculations are based. From the results obtained by direct titration and with the propionate technique, we tentatively conclude that beta i in neurons and astrocytes are approximately 20 and 30 mmol.l-1 x pH-1, respectively. We further suggest that the term "intrinsic buffer capacity", as commonly used, is redefined.

Cadmium is a toxic agent that it is also an environmental contaminant. Cadmium exposure may be implicated in some humans disorders related to hyperactivity and increased aggressiveness. This study presents data indicating that cadmium induces cellular death in corticalneurons in culture. This death could be mediated by an apoptotic and a necrotic mechanism. The apoptotic death may be mediated by oxidative stress with reactive oxygen species (ROS) formation which could be induced by mitochondrial membrane dysfunction since this cation produces: (a) depletion of mitochondrial membrane potential and (b) diminution of ATP levels with ATP release. Necrotic death could be mediated by lipid peroxidation induced by cadmium through an indirect mechanism (ROS formation). On the other hand, 40% of the cells survive cadmium action. This survival seems to be mediated by the ability of these cells to activate antioxidant defense systems, since cadmium reduced the intracellular glutathione levels and induced catalase and SOD activation in these cells.

Disruption of functional connectivity between brain regions may represent an early functional consequence of β-amyloid pathology prior to clinical Alzheimer's disease. We aimed to investigate if non-demented older individuals with increased amyloid burden demonstrate disruptions of functional whole-brain connectivity in cortical hubs (brain regions typically highly connected to multiple other brain areas) and if these disruptions are associated with neuronal dysfunction as measured with fluorodeoxyglucose-positron emission tomography. In healthy subjects without cognitive symptoms and patients with mild cognitive impairment, we used positron emission tomography to assess amyloid burden and cerebral glucose metabolism, structural magnetic resonance imaging to quantify atrophy and novel resting state functional magnetic resonance imaging processing methods to calculate whole-brain connectivity. Significant disruptions of whole-brain connectivity were found in amyloid-positive patients with mild cognitive impairment in typical cortical hubs (posterior cingulate cortex/precuneus), strongly overlapping with regional hypometabolism. Subtle connectivity disruptions and hypometabolism were already present in amyloid-positive asymptomatic subjects. Voxel-based morphometry measures indicate that these findings were not solely a consequence of regional atrophy. Whole-brain connectivity values and metabolism showed a positive correlation with each other and a negative correlation with amyloid burden. These results indicate that disruption of functional connectivity and hypometabolism may represent early functional consequences of emerging molecular Alzheimer's disease pathology, evolving prior to clinical onset of dementia. The spatial overlap between hypometabolism and disruption of connectivity in cortical hubs points to a particular susceptibility of these regions to early Alzheimer's-type neurodegeneration and may reflect a link between synaptic dysfunction and functional

3,4-Methylenedioxymethamphetamine (MDMA or "Ecstasy") is a widely abused, psychoactive recreational drug. There is growing evidence that the MDMA neurotoxic profile may be highly dependent on both its hepatic metabolism and body temperature. Metabolism of MDMA involves N-demethylation to 3,4-methylenedioxyamphetamine (MDA), which is also a drug of abuse. MDMA and MDA are O-demethylenated to N-methyl-alpha-methyldopamine (N-Me-alpha-MeDA) and alpha-methyldopamine (alpha-MeDA), respectively, both of which are catechols that can undergo oxidation to the corresponding ortho-quinones. In the presence of glutathione (GSH), ortho-quinones may be conjugated with GSH to form glutathionyl adducts. In this study, we evaluated the neurotoxicity of MDMA and three of its metabolites obtained by synthesis, N-Me-alpha-MeDA, alpha-MeDA, and 5-(GSH)-alpha-MeDA [5-(glutathion-S-yl)-alpha-methyldopamine] in rat corticalneuronal serum-free cultures under normal (36.5 degrees C) and hyperthermic (40 degrees C) conditions. Cell viability was assessed, and the mechanism of cell death was also evaluated. Our study shows that these metabolites are more neurotoxic [5-(GSH)-alpha-MeDA being the most toxic] than the parent compound MDMA. The neurotoxicity of MDMA metabolites was partially prevented by the antioxidants N-acetylcystein and also, in a minor extent, by alpha-phenyl-N-tert-butyl nitrone. All the tested compounds induced apoptotic cell death in corticalneurons, and their neurotoxic effect was potentiated under hyperthermic conditions. These data suggest that MDMA metabolites, especially under hyperthermic conditions, contribute to MDMA-induced neurotoxicity.

Full Text Available To understand how haploinsufficiency of progranulin (PGRN causes frontotemporal dementia (FTD, we created induced pluripotent stem cells (iPSCs from patients carrying the GRNIVS1+5G > C mutation (FTD-iPSCs. FTD-iPSCs were fated to corticalneurons, the cells most affected in FTD. Although generation of neuroprogenitors was unaffected, their further differentiation into CTIP2-, FOXP2-, or TBR1-TUJ1 double-positive corticalneurons, but not motorneurons, was significantly decreased in FTD-neural progeny. Zinc finger nuclease-mediated introduction of GRN cDNA into the AAVS1 locus corrected defects in cortical neurogenesis, demonstrating that PGRN haploinsufficiency causes inefficient corticalneuron generation. RNA sequencing analysis confirmed reversal of the altered gene expression profile following genetic correction. We identified the Wnt signaling pathway as one of the top defective pathways in FTD-iPSC-derived neurons, which was reversed following genetic correction. Differentiation of FTD-iPSCs in the presence of a WNT inhibitor mitigated defective corticogenesis. Therefore, we demonstrate that PGRN haploinsufficiency hampers corticogenesis in vitro.

low-dose group and ligustrazine high-dose group received ligustrazine injections, 50 mg/kg and 100 mg/kg, respectively. Samples were collected at the same time as the model group.MAIN OUTCOME MEASURES: Alterations of the neuronal ultrastructure and main organelles were ob-served by electron microscopy.RESULTS: Forty Wistar rats were included in the final analysis. Plentiful ribosome and rough endoplasmic reticulum existed in the cytoplasm of corticalneurons in the normal group. Edema existed in the nucleus and cytoplasm of neurons in the model group. The cell membrane was damaged, resulting in the external erup-tion of certain cellular organelles. In the low-dose ligustrazine group, neuronal swelling was decreased in the cytoplasm, whereas cellular organelles were relatively increased. However, the mitochondria remained swollen. The double layer structure disappeared in parts of the mitochondrial membrane. The caryotheca was still broken, and neuronal damage was significantly decreased in the high-dose ligustrazine group. In ad-dition, cytoplasmic swelling was reduced andmost part of caryotheca was complete. Fragmentation of the cellular membrane was not detected. Mitochondrial cristae and the lysosome could also be detected. The number of rough endoplasmic reticulum and free ribosomes was increased, and the structure of great part of caryotheca was clear. In addition, the number of nuclear pore was increased. However, the nuclear hetero-chromatin was relatively reduced.CONCLUSION: In the rat, the protective effects of ligustrazine were significant on neuronal membrane structures and main organelles after cerebral ischemia/reperfusion. There was a dose-dependent effect be-tween neuronal changes and Ligustrazine.

Full Text Available IL-10, as a cytokine, has an anti-inflammatory cascade following various injuries, but it remains blurred whether IL-10 protects neurites of corticalneurons after oxygen-glucose deprivation injury. Here, we reported that IL-10, in a concentration-dependent manner, reduced neuronal apoptosis and increased neuronal survival in oxygen-glucose-deprived primary corticalneurons, producing an optimal protective effect at 20ng/ml. After staining NF-H and GAP-43, we found that IL-10 significantly protected neurites in terms of axon length and dendrite number by confocal microscopy. Furthermore, it induced the phosphorylation of AKT, suppressed the activation of caspase-3, and up-regulated the protein expression of GAP-43. In contrast, LY294002, a specific inhibitor of PI3K/AKT, reduced the level of AKT phosphorylation and GAP-43 expression, increased active caspase-3 expression and thus significantly weakened IL-10-mediated protective effect in the OGD-induced injury model. IL-10NA, the IL-10 neutralizing antibody, reduced the level of p-PI3K phosphorylation and increased the expression of active caspase-3. These findings suggest that IL-10 provides neuroprotective effects by protecting neurites through PI3K/AKT signaling pathway in oxygen-glucose-deprived primary corticalneurons.

Full Text Available Phenylketonuria (PKU, an autosomal recessive disorder of amino acid metabolism caused by mutations in the phenylalanine hydroxylase (PAH gene, leads to childhood mental retardation by exposing neurons to cytotoxic levels of phenylalanine (Phe. A recent study showed that the mitochondria-mediated (intrinsic apoptotic pathway is involved in Phe-induced apoptosis in cultured corticalneurons, but it is not known if the death receptor (extrinsic apoptotic pathway and endoplasmic reticulum (ER stress-associated apoptosis also contribute to neurodegeneration in PKU. To answer this question, we used specific inhibitors to block each apoptotic pathway in corticalneurons under neurotoxic levels of Phe. The caspase-8 inhibitor Z-IETD-FMK strongly attenuated apoptosis in Phe-treated neurons (0.9 mM, 18 h, suggesting involvement of the Fas receptor (FasR-mediated cell death receptor pathway in Phe toxicity. In addition, Phe significantly increased cell surface Fas expression and formation of the Fas/FasL complex. Blocking Fas/FasL signaling using an anti-Fas antibody markedly inhibited apoptosis caused by Phe. In contrast, blocking the ER stress-induced cell death pathway with salubrinal had no effect on apoptosis in Phe-treated corticalneurons. These experiments demonstrate that the Fas death receptor pathway contributes to Phe-induced apoptosis and suggest that inhibition of the death receptor pathway may be a novel target for neuroprotection in PKU patients.

Full Text Available GABAA receptors distributed in somatodendritic compartments play critical roles in regulating neuronal activities, including spike timing and firing pattern; however, the properties and functions of GABAA receptors at the axon are still poorly understood. By recording from the cut end (bleb of the main axon trunk of layer -5 pyramidal neurons in prefrontal cortical slices, we found that currents evoked by GABA iontophoresis could be blocked by picrotoxin, indicating the expression of GABAA receptors in axons. Stationary noise analysis revealed that single-channel properties of axonal GABAA receptors were similar to those of somatic receptors. Perforated patch recording with gramicidin revealed that the reversal potential of the GABA response was more negative than the resting membrane potential at the axon trunk, suggesting that GABA may hyperpolarize the axonal membrane potential. Further experiments demonstrated that the activation of axonal GABAA receptors regulated the amplitude and duration of action potentials (APs and decreased the AP-induced Ca2+ transients at the axon. Together, our results indicate that the waveform of axonal APs and the downstream Ca2+ signals are modulated by axonal GABAA receptors.

Several findings suggest that Herpes simplex virus-1 (HSV-1) infection plays a role in the neurodegenerative processes that characterize Alzheimer's disease (AD), but the underlying mechanisms have yet to be fully elucidated. Here we show that HSV-1 productive infection in corticalneurons causes the accumulation of DNA lesions that include both single (SSBs) and double strand breaks (DSBs), which are reported to be implicated in the neuronal loss observed in neurodegenerative diseases. We demonstrate that HSV-1 downregulates the expression level of Ku80, one of the main components of non-homologous end joining (NHEJ), a major pathway for the repair of DSBs. We also provide data suggesting that HSV-1 drives Ku80 for proteasomal degradation and impairs NHEJ activity, leading to DSB accumulation. Since HSV-1 usually causes life-long recurrent infections, it is possible to speculate that cumulating damages, including those occurring on DNA, may contribute to virus induced neurotoxicity and neurodegeneration, further suggesting HSV-1 as a risk factor for neurodegenerative conditions.

The monoamine system in the prefrontal cortex has been implicated in various mental disorders and has been the major target of anxiolytics and antidepressants. Clinical studies show that serotonin and norepinephrine reuptake inhibitors (SNRIs) produce better therapeutic effects than single selective reuptake inhibitors, but the underlying mechanisms are largely unknown. Here, we found that low dose SNRIs, by acting on 5-HT(1A) and α2-adrenergic receptors, synergistically reduced AMPA receptor (AMPAR)-mediated excitatory postsynaptic currents and AMPAR surface expression in prefrontal cortex pyramidal neurons via a mechanism involving Rab5/dynamin-mediated endocytosis of AMPARs. The synergistic effect of SNRIs on AMPARs was blocked by inhibition of activator of G protein signaling 3, a G protein modulator that prevents reassociation of G(i) protein α subunit and prolongs the βγ-mediated signaling pathway. Moreover, the depression of AMPAR-mediated excitatory postsynaptic currents by SNRIs required p38 kinase activity, which was increased by 5-HT(1A) and α2-adrenergic receptor co-activation in an activator of G protein signaling 3-dependent manner. These results have revealed a potential mechanism for the synergy between the serotonin and norepinephrine systems in the regulation of glutamatergic transmission in corticalneurons.

Several findings suggest that Herpes simplex virus-1 (HSV-1) infection plays a role in the neurodegenerative processes that characterize Alzheimer’s disease (AD), but the underlying mechanisms have yet to be fully elucidated. Here we show that HSV-1 productive infection in corticalneurons causes the accumulation of DNA lesions that include both single (SSBs) and double strand breaks (DSBs), which are reported to be implicated in the neuronal loss observed in neurodegenerative diseases. We demonstrate that HSV-1 downregulates the expression level of Ku80, one of the main components of non-homologous end joining (NHEJ), a major pathway for the repair of DSBs. We also provide data suggesting that HSV-1 drives Ku80 for proteasomal degradation and impairs NHEJ activity, leading to DSB accumulation. Since HSV-1 usually causes life-long recurrent infections, it is possible to speculate that cumulating damages, including those occurring on DNA, may contribute to virus induced neurotoxicity and neurodegeneration, further suggesting HSV-1 as a risk factor for neurodegenerative conditions. PMID:27803664

Compared with the Monte Carlo method, the population density method is efficient for modeling collective dynamics of neuronal populations in human brain. In this method, a population density function describes the probabilistic distribution of states of all neurons in the population and it is governed by a hyperbolic partial differential equation. In the past, the problem was mainly solved by using the finite difference method. In a previous study, a continuous Galerkin finite element method was found better than the finite difference method for solving the hyperbolic partial differential equation; however, the population density function often has discontinuity and both methods suffer from a numerical stability problem. The goal of this study is to improve the numerical stability of the solution using discontinuous Galerkin finite element method. To test the performance of the new approach, interaction of a population of cortical pyramidal neurons and a population of thalamic neurons was simulated. The numerical results showed good agreement between results of discontinuous Galerkin finite element and Monte Carlo methods. The convergence and accuracy of the solutions are excellent. The numerical stability problem could be resolved using the discontinuous Galerkin finite element method which has total-variation-diminishing property. The efficient approach will be employed to simulate the electroencephalogram or dynamics of thalamocortical network which involves three populations, namely, thalamic reticular neurons, thalamocortical neurons and cortical pyramidal neurons.

Full Text Available Following trauma of the adult brain or spinal cord the injured axons of central neurons fail to regenerate or if intact display only limited anatomical plasticity through sprouting. Adult corticalneurons forming the corticospinal tract (CST normally have low levels of the neuronal calcium sensor-1 (NCS1 protein. In primary cultured adult corticalneurons, the lentivector-induced overexpression of NCS1 induces neurite sprouting associated with increased phospho-Akt levels. When the PI3K/Akt signalling pathway was pharmacologically inhibited the NCS1-induced neurite sprouting was abolished. The overexpression of NCS1 in uninjured corticospinal neurons exhibited axonal sprouting across the midline into the CST-denervated side of the spinal cord following unilateral pyramidotomy. Improved forelimb function was demonstrated behaviourally and electrophysiologically. In injured corticospinal neurons, overexpression of NCS1 induced axonal sprouting and regeneration and also neuroprotection. These findings demonstrate that increasing the levels of intracellular NCS1 in injured and uninjured central neurons enhances their intrinsic anatomical plasticity within the injured adult central nervous system.

Neurons of the cerebral cortex are organized in layers and columns. Unlike laminar patterning, the mechanisms underlying columnar organization remain largely unexplored. Here, we show that ephrin-B1 plays a key role in this process through the control of nonradial steps of migration of pyramidal neurons. In vivo gain of function of ephrin-B1 resulted in a reduction of tangential motility of pyramidal neurons, leading to abnormal neuronal clustering. Conversely, following genetic disruption of ephrin-B1, corticalneurons displayed a wider lateral dispersion, resulting in enlarged ontogenic columns. Dynamic analyses revealed that ephrin-B1 controls the lateral spread of pyramidal neurons by limiting neurite extension and tangential migration during the multipolar phase. Furthermore, we identified P-Rex1, a guanine-exchange factor for Rac3, as a downstream ephrin-B1 effector required to control migration during the multipolar phase. Our results demonstrate that ephrin-B1 inhibits nonradial migration of pyramidal neurons, thereby controlling the pattern of cortical columns.

Tinnitus is the phantom perception of sound in the absence of an acoustic stimulus. To date, the purported neural correlates of tinnitus from animal models have not been adequately characterized with translational technology in the human brain. The aim of the present study was to measure changes in oxy-hemoglobin concentration from regions of interest (ROI; auditory cortex) and non-ROI (adjacent nonauditory cortices) during auditory stimulation and silence in participants with subjective tinnitus appreciated equally in both ears and in nontinnitus controls using functional near-infrared spectroscopy (fNIRS). Control and tinnitus participants with normal/near-normal hearing were tested during a passive auditory task. Hemodynamic activity was monitored over ROI and non-ROI under episodic periods of auditory stimulation with 750 or 8000 Hz tones, broadband noise, and silence. During periods of silence, tinnitus participants maintained increased hemodynamic responses in ROI, while a significant deactivation was seen in controls. Interestingly, non-ROI activity was also increased in the tinnitus group as compared to controls during silence. The present results demonstrate that both auditory and select nonauditory cortices have elevated hemodynamic activity in participants with tinnitus in the absence of an external auditory stimulus, a finding that may reflect basic science neural correlates of tinnitus that ultimately contribute to phantom sound perception.

Full Text Available Tinnitus is the phantom perception of sound in the absence of an acoustic stimulus. To date, the purported neural correlates of tinnitus from animal models have not been adequately characterized with translational technology in the human brain. The aim of the present study was to measure changes in oxy-hemoglobin concentration from regions of interest (ROI; auditory cortex and non-ROI (adjacent nonauditory cortices during auditory stimulation and silence in participants with subjective tinnitus appreciated equally in both ears and in nontinnitus controls using functional near-infrared spectroscopy (fNIRS. Control and tinnitus participants with normal/near-normal hearing were tested during a passive auditory task. Hemodynamic activity was monitored over ROI and non-ROI under episodic periods of auditory stimulation with 750 or 8000 Hz tones, broadband noise, and silence. During periods of silence, tinnitus participants maintained increased hemodynamic responses in ROI, while a significant deactivation was seen in controls. Interestingly, non-ROI activity was also increased in the tinnitus group as compared to controls during silence. The present results demonstrate that both auditory and select nonauditory cortices have elevated hemodynamic activity in participants with tinnitus in the absence of an external auditory stimulus, a finding that may reflect basic science neural correlates of tinnitus that ultimately contribute to phantom sound perception.

The present quantitative study extends our investigation of cetartiodactyls by exploring the neuronal morphology in the giraffe (Giraffa camelopardalis) neocortex. Here, we investigate giraffe primary visual and motor cortices from perfusion-fixed brains of three subadults stained with a modified rapid Golgi technique. Neurons (n = 244) were quantified on a computer-assisted microscopy system. Qualitatively, the giraffe neocortex contained an array of complex spiny neurons that included both "typical" pyramidal neuron morphology and "atypical" spiny neurons in terms of morphology and/or orientation. In general, the neocortex exhibited a vertical columnar organization of apical dendrites. Although there was no significant quantitative difference in dendritic complexity for pyramidal neurons between primary visual (n = 78) and motor cortices (n = 65), there was a significant difference in dendritic spine density (motor cortex > visual cortex). The morphology of aspiny neurons in giraffes appeared to be similar to that of other eutherian mammals. For cross-species comparison of neuron morphology, giraffe pyramidal neurons were compared to those quantified with the same methodology in African elephants and some cetaceans (e.g., bottlenose dolphin, minke whale, humpback whale). Across species, the giraffe (and cetaceans) exhibited less widely bifurcating apical dendrites compared to elephants. Quantitative dendritic measures revealed that the elephant and humpback whale had more extensive dendrites than giraffes, whereas the minke whale and bottlenose dolphin had less extensive dendritic arbors. Spine measures were highest in the giraffe, perhaps due to the high quality, perfusion fixation. The neuronal morphology in giraffe neocortex is thus generally consistent with what is known about other cetartiodactyls.

The present study utilizes nestin-BDNF transgenic mice, which offer a model for early increased brain-derived neurotrophic factor (BDNF) signalling, to examine the role of BDNF in the development of cortical architecture. Our results demonstrate that the premature and homogeneous expression of BDNF, while preserving tangential migration from the ganglionic eminence to the cortex, impairs the final radial migration of GABAergic neurons, as well as their integration in the appropriate cortical layers. Moreover, Cajal-Retzius (CR) cells and GABAergic neurons segregate in the cortical marginal zone (MZ) in response to BDNF signalling, leading to an alternating pattern and a columnar cortical organization, within which the migration of different neuronal populations is specifically affected. These results suggest that both CR and GABAergic neurons play a role in directing the radial migration of late-generated corticalneurons, and that the spatial distribution of these cells in the MZ is critical for the development of correct cortical organization. In addition, reelin secreted by CR cells in the MZ is not sufficient to direct the migration of late-born neurons to the upper cortical layers, which most likely requires the presence of reelin-secreting interneurons in layers V-VI. We propose that in addition to modulating reelin expression, BDNF regulates the patched distribution of CR and GABAergic neurons in the MZ, and that this spatial distribution is involved in the formation of anatomical and/or functional columns and convoluted structures.

Full Text Available The brain is considered to use a relatively small amount of energy for its efficient information processing. Under a severe restriction on the energy consumption, the maximization of mutual information (MMI, which is adequate for designing artificial processing machines, may not suit for the brain. The MMI attempts to send information as accurate as possible and this usually requires a sufficient energy supply for establishing clearly discretized communication bands. Here, we derive an alternative hypothesis for neural code from the neuronal activities recorded juxtacellularly in the sensorimotor cortex of behaving rats. Our hypothesis states that in vivo corticalneurons maximize the entropy of neuronal firing under two constraints, one limiting the energy consumption (as assumed previously and one restricting the uncertainty in output spike sequences at given firing rate. Thus, the conditional maximization of firing-rate entropy (CMFE solves a tradeoff between the energy cost and noise in neuronal response. In short, the CMFE sends a rich variety of information through broader communication bands (i.e., widely distributed firing rates at the cost of accuracy. We demonstrate that the CMFE is reflected in the long-tailed, typically power law, distributions of inter-spike intervals obtained for the majority of recorded neurons. In other words, the power-law tails are more consistent with the CMFE rather than the MMI. Thus, we propose the mathematical principle by which corticalneurons may represent information about synaptic input into their output spike trains.

Histone deacetylase inhibitors (HDACi)-valproic acid (VPA) and trichostatin A (TSA) promote neurogenesis, neurite outgrowth, synaptic plasticity and neuroprotection. In this study, we investigated whether VPA and TSA promote post-ischemic neuroprotection and neuronal restoration in rat primary corticalneurons. On 6 days in vitro (DIV), corticalneurons were exposed to oxygen-glucose deprivation for 90 min. Cells were returned to normoxic conditions and cultured for 1, 3, or 7 days with or without VPA and TSA. Control cells were cultured in normoxic conditions only. On 7, 9, and 13 DIV, cells were measured neurite outgrowth using the Axiovision program and stained with Tunel staining kit. Microtubule associated protein-2 immunostaining and tunel staining showed significant recovery of neurite outgrowth and post-ischemic neuronal death by VPA or TSA treatment. We also determined levels of acetylated histone H3, PSD95, GAP 43 and synaptophysin. Significant increases in all three synaptic markers and acetylated histone H3 were observed relative to non-treated cells. Post-ischemic HDACi treatment also significantly raised levels of brain derived neurotrophic factor (BDNF) expression and secreted BDNF. Enhanced BDNF expression by HDACi treatment might have been involved in the post-ischemic neuroprotection and neuronal restorative effects. Our findings suggest that both VPA and TSA treatment during reoxygenation after ischemia may help post-ischemic neuroprotection and neuronal regeneration via increased BDNF expression and activation.

Prenatal ethanol exposure disrupts cortical neurite initiation and outgrowth, but prior studies have reported both ethanol-dependent growth promotion and inhibition. To resolve this ambiguity and better approximate in vivo conditions, we quantitatively analyzed neuronal morphology using a new, whole hemisphere explant model. In this model, Layer 6 (L6) corticalneurons migrate, laminate and extend neurites in an organotypic fashion. To selectively label L6 neurons, we performed ex utero electroporation of a GFP expression construct at embryonic day 13 and allowed the explants to develop for 2 days in vitro. Explants were exposed to (400 mg/dL) ethanol for either 4 or 24 h prior to fixation. Complete 3-D reconstructions were made of >80 GFP-positive neurons in each experimental condition. Acute responses to ethanol exposure included compaction of the Golgi apparatus accompanied by elaboration of supernumerary primary apical neurites, as well as a modest (∼15%) increase in higher order apical neurite length. With longer exposure time, ethanol exposure leads to a consistent, significant disorientation of the cell (cell body, primary apical neurite, and Golgi) with respect to the pial surface. The effects on cellular orientation were accompanied by decreased expression of cytoskeletal elements, microtubule-associated protein 2 and F-actin. These findings indicate that upon exposure to ethanol, developing L6 neurons manifest disruptions in Golgi apparatus and cytoskeletal elements which may in turn trigger selective and significant perturbations to primary neurite formation and neuronal polarity.

In neuropathology research, induced pluripotent stem cell (iPSC)-derived neurons are considered a tool closely resembling the patient brain. Albeit in respect to epigenetics, this concept has been challenged. We generated iPSC-derived corticalneurons from myoclonus-dystonia patients with mutations (W100G and R102X) in the maternally imprinted ε-sarcoglycan (SGCE) gene and analysed properties such as imprinting, mRNA and protein expression. Comparison of the promoter during reprogramming and differentiation showed tissue-independent differential methylation. DNA sequencing with methylation-specific primers and cDNA analysis in patient neurons indicated selective expression of the mutated paternal SGCE allele. While fibroblasts only expressed the ubiquitous mRNA isoform, brain-specific SGCE mRNA and ε-sarcoglycan protein were detected in iPSC-derived control neurons. However, neuronal protein levels were reduced in both mutants. Our phenotypic characterization highlights the suitability of iPSC-derived corticalneurons with SGCE mutations for myoclonus-dystonia research and, in more general terms, prompts the use of iPSC-derived cellular models to study epigenetic mechanisms impacting on health and disease. PMID:28155872

Full Text Available Causes of lower induction of Hsp70 in neurons during heat shock are still a matter of debate. To further inquire into the mechanisms regulating Hsp70 expression in neurons, we studied the activity of Heat Shock Factor 1 (HSF1 and histone posttranslational modifications (PTMs at the hsp70 promoter in rat corticalneurons. Heat shock induced a transient and efficient translocation of HSF1 to neuronal nuclei. However, no binding of HSF1 at the hsp70 promoter was detected while it bound to the hsp25 promoter in corticalneurons during heat shock. Histone PTMs analysis showed that the hsp70 promoter harbors lower levels of histone H3 and H4 acetylation in corticalneurons compared to PC12 cells under basal conditions. Transcriptomic profiling data analysis showed a predominant usage of cryptic transcriptional start sites at hsp70 gene in the rat cerebral cortex, compared with the whole brain. These data support a weaker activation of hsp70 canonical promoter. Heat shock increased H3Ac at the hsp70 promoter in PC12 cells, which correlated with increased Hsp70 expression while no modifications occurred at the hsp70 promoter in corticalneurons. Increased histone H3 acetylation by Trichostatin A led to hsp70 mRNA and protein induction in corticalneurons. In conclusion, we found that two independent mechanisms maintain a lower induction of Hsp70 in corticalneurons. First, HSF1 fails to bind specifically to the hsp70 promoter in corticalneurons during heat shock and, second, the hsp70 promoter is less accessible in neurons compared to non-neuronal cells due to histone deacetylases repression.

Stress signaling in response to oxygen/glucose deprivation (OGD) and ischemic injury activates a group of pro-apoptotic genes, the Bcl-2 homology domain 3 (BH3)-only proteins, which are capable of activating the mitochondrial apoptosis pathway. Targeted studies previously identified the BH3-only proteins Puma, Bim and Bid to have a role in ischemic/hypoxic neuronal injury. We here investigated the transcriptional activation of pro-apoptotic BH3-only proteins after OGD-induced injury in murine neocortical neurons. We observed a potent and early upregulation of noxa at mRNA and protein level, and a significant increase in Bmf protein levels during OGD in neocortical neurons and in the ipsilateral cortex of mice subjected to transient middle cerebral artery occlusion (tMCAO). Surprisingly, gene deficiency in noxa reduced neither OGD- nor glutamate-induced neuronal injury in corticalneurons and failed to influence infarct size or neurological deficits after tMCAO. In contrast, bmf deficiency induced significant protection against OGD- or glutamate-induced injury in cultured neurons, and bmf-deficient mice showed reduced neurological deficits after tMCAO in vivo. Collectively, our data not only point to a role of Bmf as a BH3-only protein contributing to excitotoxic and ischemic neuronal injury but also demonstrate that the early and potent induction of noxa does not influence ischemic neuronal injury.

Exposure of the central nervous system to organophosphorus (OP) nerve agents induces seizures and neuronal cell death. Here we report that the OP nerve agent, VX, induces apoptotic-like cell death in cultured rat corticalneurons. The VX effects on neurons were concentration-dependent, with an IC(50) of approximately 30 microM. Blockade of N-methyl-D-aspartate receptors (NMDAR) with 50 microM. D-2-amino-5-phosphonovalerate (APV) diminished 30 microM VX-induced total cell death, as assessed by alamarBlue assay and Hoechst staining. In contrast, neither antagonists of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptors (AMPARs) nor metabotropic glutamate receptors (mGluRs) had any effect on VX-induced neurotoxicity. VX-induced neuronal cell death could not be solely attributed to acetylcholinesterase (AChE) inhibition, since neither the reversible pharmacological cholinesterase inhibitor, physostigmine, nor the muscarinic receptor antagonist, atropine, affected VX-induced cell death. Importantly, APV was found to be therapeutically effective against VX-induced cell death up to 2 h post VX exposure. These results suggest that NMDARs, but not AMPARs or mGluRs, play important roles in VX-induced cell death in cultured rat corticalneurons. Based on their therapeutic effects, NMDAR antagonists may be beneficial in the treatment of VX-induced neurotoxicities.

Various types of neural-based signals, such as EEG, local field potentials and intracellular synaptic potentials, integrate multiple sources of activity distributed across large assemblies. They have in common a power-law frequency-scaling structure at high frequencies, but it is still unclear whether this scaling property is dominated by intrinsic neuronal properties or by network activity. The latter case is particularly interesting because if frequency-scaling reflects the network state it could be used to characterize the functional impact of the connectivity. In intracellularly recorded neurons of cat primary visual cortex in vivo, the power spectral density of V(m) activity displays a power-law structure at high frequencies with a fractional scaling exponent. We show that this exponent is not constant, but depends on the visual statistics used to drive the network. To investigate the determinants of this frequency-scaling, we considered a generic recurrent model of cortex receiving a retinotopically organized external input. Similarly to the in vivo case, our in computo simulations show that the scaling exponent reflects the correlation level imposed in the input. This systematic dependence was also replicated at the single cell level, by controlling independently, in a parametric way, the strength and the temporal decay of the pairwise correlation between presynaptic inputs. This last model was implemented in vitro by imposing the correlation control in artificial presynaptic spike trains through dynamic-clamp techniques. These in vitro manipulations induced a modulation of the scaling exponent, similar to that observed in vivo and predicted in computo. We conclude that the frequency-scaling exponent of the V(m) reflects stimulus-driven correlations in the cortical network activity. Therefore, we propose that the scaling exponent could be used to read-out the "effective" connectivity responsible for the dynamical signature of the population signals measured

Full Text Available Various types of neural-based signals, such as EEG, local field potentials and intracellular synaptic potentials, integrate multiple sources of activity distributed across large assemblies. They have in common a power-law frequency-scaling structure at high frequencies, but it is still unclear whether this scaling property is dominated by intrinsic neuronal properties or by network activity. The latter case is particularly interesting because if frequency-scaling reflects the network state it could be used to characterize the functional impact of the connectivity. In intracellularly recorded neurons of cat primary visual cortex in vivo, the power spectral density of V(m activity displays a power-law structure at high frequencies with a fractional scaling exponent. We show that this exponent is not constant, but depends on the visual statistics used to drive the network. To investigate the determinants of this frequency-scaling, we considered a generic recurrent model of cortex receiving a retinotopically organized external input. Similarly to the in vivo case, our in computo simulations show that the scaling exponent reflects the correlation level imposed in the input. This systematic dependence was also replicated at the single cell level, by controlling independently, in a parametric way, the strength and the temporal decay of the pairwise correlation between presynaptic inputs. This last model was implemented in vitro by imposing the correlation control in artificial presynaptic spike trains through dynamic-clamp techniques. These in vitro manipulations induced a modulation of the scaling exponent, similar to that observed in vivo and predicted in computo. We conclude that the frequency-scaling exponent of the V(m reflects stimulus-driven correlations in the cortical network activity. Therefore, we propose that the scaling exponent could be used to read-out the "effective" connectivity responsible for the dynamical signature of the population

Full Text Available Neocortex functioning relies on the formation of complex networks that begins to be assembled during embryogenesis by highly stereotyped processes of cell migration and axonal navigation. The guidance of cells and axons is driven by extracellular cues, released along by final targets or intermediate targets located along specific pathways. In particular, guidepost cells, originally described in the grasshopper, are considered discrete, specialized cell populations located at crucial decision points along axonal trajectories that regulate tract formation. These cells are usually early-born, transient and act at short-range or via cell-cell contact. The vast majority of guidepost cells initially identified were glial cells, which play a role in the formation of important axonal tracts in the forebrain, such as the corpus callosum, anterior and post-optic commissures as well as optic chiasm. In the last decades, tangential migrating neurons have also been found to participate in the guidance of principal axonal tracts in the forebrain. This is the case for several examples such as guideposts for the lateral olfactory tract (LOT, corridor cells, which open an internal path for thalamo-cortical axons and Cajal-Retzius cells that have been involved in the formation of the entorhino-hippocampal connections. More recently, microglia, the resident macrophages of the brain, were specifically observed at the crossroads of important neuronal migratory routes and axonal tract pathways during forebrain development. We furthermore found that microglia participate to the shaping of prenatal forebrain circuits, thereby opening novel perspectives on forebrain development and wiring. Here we will review the last findings on already known guidepost cells populations and will discuss the role of microglia as a potentially new class of atypical guidepost cells.

Cortical spreading depression is associated with activation of NMDA receptors, which interact with the postsynaptic density protein 95 (PSD-95) that binds to nitric oxide synthase (nNOS). Here, we tested whether inhibition of the nNOS/PSD-95/NMDA receptor complex formation by anti-ischemic compound, UCCB01-144 (Tat- N-dimer) ameliorates the persistent effects of cortical spreading depression on cortical function. Using in vivo two-photon microscopy in somatosensory cortex in mice, we show that fluorescently labelled Tat- N-dimer readily crosses blood-brain barrier and accumulates in nerve cells during the first hour after i.v. injection. The Tat- N-dimer suppressed stimulation-evoked synaptic activity by 2-20%, while cortical blood flow and cerebral oxygen metabolic (CMRO2) responses were preserved. During cortical spreading depression, the Tat- N-dimer reduced the average amplitude of the negative shift in direct current potential by 33% (4.1 mV). Furthermore, the compound diminished the average depression of spontaneous electrocorticographic activity by 11% during first 40 min of post-cortical spreading depression recovery, but did not mitigate the suppressing effect of cortical spreading depression on cortical blood flow and CMRO2. We suggest that uncoupling of PSD-95 from NMDA receptors reduces overall neuronal excitability and the amplitude of the spreading depolarization wave. These findings may be of interest for understanding the neuroprotective effects of the nNOS/PSD-95 uncoupling in stroke.

Brain imaging techniques that use vascular signals to map changes in neuronal activity, such as blood oxygenation level-dependent functional magnetic resonance imaging, rely on the spatial and temporal coupling between changes in neurophysiology and haemodynamics, known as 'neurovascular coupling (NVC)'. Accordingly, NVC responses, mapped by changes in brain haemodynamics, have been validated for different stimuli under physiological conditions. In the cerebral cortex, the networks of excitatory pyramidal cells and inhibitory interneurons generating the changes in neural activity and the key mediators that signal to the vascular unit have been identified for some incoming afferent pathways. The neural circuits recruited by whisker glutamatergic-, basal forebrain cholinergic- or locus coeruleus noradrenergic pathway stimulation were found to be highly specific and discriminative, particularly when comparing the two modulatory systems to the sensory response. However, it is largely unknown whether or not NVC is still reliable when brain states are altered or in disease conditions. This lack of knowledge is surprising since brain imaging is broadly used in humans and, ultimately, in conditions that deviate from baseline brain function. Using the whisker-to-barrel pathway as a model of NVC, we can interrogate the reliability of NVC under enhanced cholinergic or noradrenergic modulation of cortical circuits that alters brain states.This article is part of the themed issue 'Interpreting BOLD: a dialogue between cognitive and cellular neuroscience'.

Fungicides are crucial for food protection as well as for the production of crops of suitable quality and quantity to provide a viable economic return. Like other pesticides, fungicides are widely sprayed on agricultural land, especially in wine-growing areas, from where they can move-off after application. Furthermore, residues of these agrochemicals can remain on crops after harvest and even after some food processing operations, being a major exposure pathway. Although a relatively low toxicity has been claimed for this kind of compounds, information about their neurotoxicity is still scarce. In the present study, nine fungicides recently approved for agricultural uses in the EU - ametoctradin, boscalid, cyazofamid, dimethomorph, fenhexamid, kresoxim-methyl, mepanipyrim, metrafenone and pyraclostrobin - have been evaluated for their toxicity in primary cultured mouse corticalneurons. Exposure to 0.1-100µM for 7 days in vitro resulted in a dose-dependent toxicity in the MTT cell viability assay. Strobilurin fungicides kresoxim-methyl (KR) and pyraclostrobin (PY) were the most neurotoxic compounds (lethal concentration 50 were in the low micromolar and nanomolar levels, respectively) causing a rapid raise in intracellular calcium [Ca(2+)]i and strong depolarization of mitochondrial membrane potential. KR- and PY-induced cell death was reversed by the calcium channels blockers MK-801 and verapamil, suggesting that calcium entry through NMDA receptors and voltage-operated calcium channels are involved in KR- and PY-induced neurotoxicity. These results highlight the need for further evaluation of their neurotoxic effects in vivo.

Full Text Available The recent conceptual achievement that the cortical motor system plays a crucial role not only in motor control but also in higher cognitive functions has given a new perspective also on the involvement of motor cortex in language perception and production. In particular, there is evidence that the matching mechanism based on mirror neurons can be involved in both pho-nological recognition and retrieval of meaning, especially for action word categories, thus suggesting a contribution of an action–perception mechanism to the automatic comprehension of semantics. Furthermore, a compari-son of the anatomo-functional properties of the frontal motor cortex among different primates and their communicative modalities indicates that the combination of the voluntary control of the gestural communication systems and of the vocal apparatus has been the critical factor in the transition from a gestural-based communication into a predominantly speech-based system. Finally, considering that the monkey and human premotor-parietal motor system, plus the prefrontal cortex, are involved in the sequential motor organization of actions and in the hierarchical combination of motor elements, we propose that elements of such motor organization have been exploited in other domains, including some aspects of the syntactic structure of language.

After CNS injury, astrocytes and mesenchymal cells attempt to restore the disrupted glia limitans by secreting proteoglycans and extracellular matrix proteins (ECMs), forming the so-called glial scar. Although the glial scar is important in sealing the lesion, it is also a physical and functional barrier that prevents axonal regeneration. The synthesis of secretory proteins in the RER is under the control of the initiation factor of translation eIF2α. Inhibiting the synthesis of secretory proteins by increasing the phosphorylation of eIF2α, might be a pharmacologically efficient way of reducing proteoglycans and other profibrotic proteins present in the glial scar. Salubrinal, a neuroprotective drug, decreased the expression and secretion of proteoglycans and other profibrotic proteins induced by EGF or TGFβ, maintaining eIF2α phosphorylated. Besides, Salubrinal also reduced the transcription of proteoglycans and other profibrotic proteins, suggesting that it induced the degradation of non-translated mRNA. In a model in vitro of the glial scar, corticalneurons grown on cocultures of astrocytes and fibroblasts with TGFβ treated with Salubrinal, showed increased neurite outgrowth compared to untreated cells. Our results suggest that Salubrinal may be considered of therapeutic value facilitating axonal regeneration, by reducing overproduction and secretion of proteoglycans and profibrotic protein inhibitors of axonal growth.

The lateral posterior thalamic nucleus (LP) is one of the components of the extrageniculate pathway in the rat visual system, and is cytoarchitecturally divided into three subdivisions--lateral (LPl), rostromedial (LPrm), and caudomedial (LPcm) portions. To clarify the differences in the dendritic fields and axonal arborisations among the three subdivisions, we applied a single-neuron labeling technique with viral vectors to LP neurons. The proximal dendrites of LPl neurons were more numerous than those of LPrm and LPcm neurons, and LPrm neurons tended to have wider dendritic fields than LPl neurons. We then analysed the axonal arborisations of LP neurons by reconstructing the axon fibers in the cortex. The LPl, LPrm and LPcm were different from one another in terms of the projection targets--the main target cortical regions of LPl and LPrm neurons were the secondary and primary visual areas, whereas those of LPcm neurons were the postrhinal and temporal association areas. Furthermore, the principal target cortical layers of LPl neurons in the visual areas were middle layers, but that of LPrm neurons was layer 1. This indicates that LPl and LPrm neurons can be categorised into the core and matrix types of thalamic neurons, respectively, in the visual areas. In addition, LPl neurons formed multiple axonal clusters within the visual areas, whereas the fibers of LPrm neurons were widely and diffusely distributed. It is therefore presumed that these two types of neurons play different roles in visual information processing by dual thalamocortical innervation of the visual areas.

Interleukin-18 (IL-18), a member of the IL-1 family of cytokines, was initially identified as an interferon (IFN)-γ-inducing factor. IL-18 is expressed in both immune and non-immune cells and participates in the adjustment of multitude cellular functions. Nonetheless, the effects of IL-18 on corticalneurons have not been explored. The present study was conducted to investigate the influence of IL-18 on rat primary corticalneurons and elucidate the underlying mechanisms. We proved that rrIL-18 increased the brain-derived neurotrophic factor (BDNF) expression in a time-dependent manner. Treatment with rrIL-18 (50 ng/ml) deactivated phosphatase and tensin homolog deleted on chromosome 10 (PTEN) by facilitating its phosphorylation, enhanced the expression of Phosphoinositide 3-OH kinase (PI3K) and p-Akt, standing for the activation of the PI3K/Akt pathway. As its pivotal downstream pathways, nuclear factor-kappa B (NF-κB), cAMP-responsive element binding protein (CREB)/Bcl-2 and glycogen synthase kinase-3β (GSK-3β) were examined in further steps. Our data revealed that rrIL-18 stimulated NF-κB activation, improved p-CREB and anti-apoptotic Bcl-2 expression levels. But rrIL-18 had little or no effect on GSK-3β pathway. Besides, rrIL-18 increased levels of BDNF and Bcl-2/Bax ratio and decreased cleaved caspase-3 expression to protect corticalneurons from damage induced by oxygen-glucose deprivation (OGD). These results in vitro showed the protection of IL-18 on corticalneurons. And this direct neuroprotective effect of IL-18 is crippled by PI3K inhibitor wortmannin.

Green tea polyphenols are a natural product which has antioxidative and antiapoptotic effects. It has been shown that glutamate excitotoxicity induced oxidative stress is linked to neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s disease. In this study we explored the neuroprotective effect of green teen polyphenols against glutamate excitotoxicity in the primary cultured corticalneurons. We found that green tea polyphenols protected against glutamate induced neurotox...

Full Text Available Abstract Background Neuroprotective and neurotrophic properties of leukemia inhibitory factor (LIF have been widely reported. In the central nervous system (CNS, astrocytes are the major source for LIF, expression of which is enhanced following disturbances leading to neuronal damage. How astrocytic LIF expression is regulated, however, has remained an unanswered question. Since neuronal stress is associated with production of extracellular adenosine, we investigated whether LIF expression in astrocytes was mediated through adenosine receptor signaling. Methods Mouse corticalneuronal and astrocyte cultures from wild-type and adenosine A2B receptor knock-out animals, as well as adenosine receptor agonists/antagonists and various enzymatic inhibitors, were used to study LIF expression and release in astrocytes. When needed, a one-way analysis of variance (ANOVA followed by Bonferroni post-hoc test was used for statistical analysis. Results We show here that glutamate-stressed corticalneurons induce LIF expression through activation of adenosine A2B receptor subtype in cultured astrocytes and require signaling of protein kinase C (PKC, mitogen-activated protein kinases (MAPKs: p38 and ERK1/2, and the nuclear transcription factor (NF-κB. Moreover, LIF concentration in the supernatant in response to 5′-N-ethylcarboxamide (NECA stimulation was directly correlated to de novo protein synthesis, suggesting that LIF release did not occur through a regulated release pathway. Immunocytochemistry experiments show that LIF-containing vesicles co-localize with clathrin and Rab11, but not with pHogrin, Chromogranin (CgA and CgB, suggesting that LIF might be secreted through recycling endosomes. We further show that pre-treatment with supernatants from NECA-treated astrocytes increased survival of cultured corticalneurons against glutamate, which was absent when the supernatants were pre-treated with an anti-LIF neutralizing antibody. Conclusions

Maturation of electrical excitability during early postnatal development is critical to formation of functional neural circuitry in the mammalian neocortex. Little is known, however, about the changes in gene expression underlying the development of firing properties that characterize different classes of corticalneurons. Here we describe the development of corticalneurons with two distinct firing phenotypes, regular-spiking (RS) and fast-spiking (FS), that appear to emerge from a populatio...

14,15-Epoxyeicosatrienoic acid (14,15-EET), a metabolite of arachidonic acid, is enriched in the brain cortex and exerts protective effect against neuronal apoptosis induced by ischemia/reperfusion. Although apoptosis has been well recognized to be closely associated with mitochondrial biogenesis and function, it is still unclear whether the neuroprotective effect of 14,15-EET is mediated by promotion of mitochondrial biogenesis and function in corticalneurons under the condition of oxygen-glucose deprivation (OGD). In this study, we found that 14,15-EET improved cell viability and inhibited apoptosis of corticalneurons. 14,15-EET significantly increased the mitochondrial mass and the ratio of mitochondrial DNA to nuclear DNA. Key makers of mitochondrial biogenesis, peroxisome proliferator activator receptor gamma-coactivator 1 alpha (PGC-1α), nuclear respiratory factor 1 (NRF-1) and mitochondrial transcription factor A (TFAM), were elevated at both mRNA and protein levels in the corticalneurons treated with 14,15-EET. Moreover, 14,15-EET markedly attenuated the decline of mitochondrial membrane potential, reduced ROS, while increased ATP synthesis. Knockdown of cAMP-response element binding protein (CREB) by siRNA blunted the up-regulation of PGC-1α and NRF-1 stimulated by 14,15-EET, and consequently abolished the neuroprotective effect of 14,15-EET. Our results indicate that 14,15-EET protects neurons from OGD-induced apoptosis by promoting mitochondrial biogenesis and function through CREB mediated activation of PGC-1α and NRF-1.

Correlation between cortical activity and electromyographic (EMG) activity of limb muscles has long been a subject of neurophysiological studies, especially in terms of corticospinal connectivity. Interest in this issue has recently increased due to the development of brain-machine interfaces with output signals that mimic muscle force. For this study, three monkeys were implanted with multielectrode arrays in multiple cortical areas. One monkey performed self-timed touch pad presses, whereas the other two executed arm reaching movements. We analyzed the dynamic relationship between corticalneuronal activity and arm EMGs using a joint cross-correlation (JCC) analysis that evaluated trial-by-trial correlation as a function of time intervals within a trial. JCCs revealed transient correlations between the EMGs of multiple muscles and neural activity in motor, premotor and somatosensory cortical areas. Matching results were obtained using spike-triggered averages corrected by subtracting trial-shuffled data. Compared with spike-triggered averages, JCCs more readily revealed dynamic changes in cortico-EMG correlations. JCCs showed that correlation peaks often sharpened around movement times and broadened during delay intervals. Furthermore, JCC patterns were directionally selective for the arm-reaching task. We propose that such highly dynamic, task-dependent and distributed relationships between cortical activity and EMGs should be taken into consideration for future brain-machine interfaces that generate EMG-like signals. PMID:25210153

Full Text Available Proper density and morphology of dendritic spines are important for higher brain functions such as learning and memory. However, our knowledge about molecular mechanisms that regulate the development and maintenance of dendritic spines is limited. We recently reported that cyclin-dependent kinase 5 (Cdk5 is required for the development and maintenance of dendritic spines of corticalneurons in the mouse brain. Previous in vitro studies have suggested the involvement of Cdk5 substrates in the formation of dendritic spines; however, their role in spine development has not been tested in vivo. Here, we demonstrate that Cdk5 phosphorylates collapsin response mediator protein 2 (CRMP2 in the dendritic spines of cultured hippocampal neurons and in vivo in the mouse brain. When we eliminated CRMP2 phosphorylation in CRMP2KI/KI mice, the densities of dendritic spines significantly decreased in hippocampal CA1 pyramidal neurons in the mouse brain. These results indicate that phosphorylation of CRMP2 by Cdk5 is important for dendritic spine development in corticalneurons in the mouse hippocampus.

Full Text Available Green tea polyphenols are a natural product which has antioxidative and antiapoptotic effects. It has been shown that glutamate excitotoxicity induced oxidative stress is linked to neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s disease. In this study we explored the neuroprotective effect of green teen polyphenols against glutamate excitotoxicity in the primary cultured corticalneurons. We found that green tea polyphenols protected against glutamate induced neurotoxicity in the corticalneurons as measured by MTT and TUNEL assays. Green tea polyphenols were then showed to inhibit the glutamate induced ROS release and SOD activity reduction in the neurons. Furthermore, our results demonstrated that green tea polyphenols restored the dysfunction of mitochondrial pro- or antiapoptotic proteins Bax, Bcl-2, and caspase-3 caused by glutamate. Interestingly, the neuroprotective effect of green tea polyphenols was abrogated when the neurons were incubated with siBcl-2. Taken together, these results demonstrated that green tea polyphenols protected against glutamate excitotoxicity through antioxidative and antiapoptotic pathways.

Large research activity has raised around the mechanisms of interaction between extremely low-frequency magnetic fields (ELF-MFs) and biological systems. ELF-MFs may interfere with chemical reactions involving reactive oxygen species (ROS), thus facilitating oxidative damages in living cells. Corticalneurons are particularly susceptible to oxidative stressors and are also highly dependent on the specific factors and proteins governing neuronal development, activity and survival. The aim of the present work was to investigate the effects of exposures to two different 50 Hz sinusoidal ELF-MFs intensities (0.1 and 1 mT) in maturing rat corticalneurons' major anti-oxidative enzymatic and non-enzymatic cellular protection systems, membrane peroxidative damage, as well as growth factor, and cytokine expression pattern. Briefly, our results showed that ELF-MFs affected positively the cell viability and concomitantly reduced the levels of apoptotic death in rat neuronal primary cultures, with no significant effects on the main anti-oxidative defences. Interestingly, linear regression analysis suggested a positive correlation between reduced glutathione (GSH) and ROS levels in 1 mT MF-exposed cells. On this basis, our hypothesis is that GSH could play an important role in the antioxidant defence towards the ELF-MF-induced redox challenge. Moreover, the GSH-based cellular response was achieved together with a brain-derived neurotrophic factor over-expression as well as with the interleukin 1beta-dependent regulation of pro-survival signaling pathways after ELF-MF exposure.

Full Text Available In the murine brain, the first post-mitotic corticalneurons formed during embryogenesis express store-operated channels (SOCs sensitive to Pyr3, initially proposed as a blocker of transient receptor potential channel of C type 3 (TRPC3 channel. However Pyr3 does not discriminate between Orai and TRPC3 channels, questioning the contribution of TRPC3 in SOCs. This study was undertaken to precise the molecular identity and the pharmacological profile of native SOCs from E13 corticalneurons. The mRNA expression of STIM1-2, Orai1-3 was assessed by quantitative reverse transcription polymerase chain reaction (RT-PCR. E13 corticalneurons expressed STIM1-2 mRNAs, with STIM2 being the predominant isoform. Only transcripts of Orai2 were found but no Orai1 and Orai3 mRNAs. Blockers of Orai and TRPC channels (Pyr6, Pyr10, EVP4593, SAR7334, GSK-7975A were used to further characterize the endogenous SOCs. Their activity was recorded using the fluorescent Ca2+ probe Fluo-4. Cortical SOCs were sensitive to the Orai blockers Pyr6, GSK-7975A, and also to EVP4593, zinc, copper and gadolinium ions, the latter one being the most potent SOCs blocker tested (IC50 ~10 nM. SOCs were insensitive to the TRPC channel blockers Pyr10 and SAR7334. In addition, preventing the mitochondrial Ca2+ uptake inhibited SOCs which were unaffected by inhibitors of the Ca2+-independent phospholipase A2 (iPLA2. Altogether, Orai2 channels are present at the beginning of the embryonic murine cortico-genesis and form the core component of native SOCs in the immature cortex. This Ca2+ route is likely to play a role in the formation of the brain cortex.

Trans-synaptic interaction of postsynaptic glutamate receptor δ2 and presynaptic neurexins (NRXNs) through cerebellin precursor protein (Cbln) 1 mediates synapse formation in the cerebellum [T. Uemura, S.J. Lee, M. Yasumura, T. Takeuchi, T. Yoshida, M. Ra, R. Taguchi, K. Sakimura, M. Mishina, Cell 141 (2010) 1068-1079]. This finding raises a question whether other Cbln family members interact with NRXNs to regulate synapse formation in the forebrain. Here, we showed that Cbln1 and Cbln2 induced presynaptic differentiation of cultured corticalneurons, while Cbln4 exhibited little activity. When compared with neuroligin 1, Cbln1 and Cbln2 induced preferentially inhibitory presynaptic differentiation rather than excitatory one in cortical cultures. The synaptogenic activities of Cbln1 and Cbln2 were suppressed by the addition of the extracellular domain of NRXN1β to the corticalneuron cultures. Consistently, Cbln1 and Cbln2 showed robust binding activities to NRXN1α and three β-NRXNs, while only weak interactions were observed between Cbln4 and NRXNs. The interactions of Cbln1, Cbln2 and Cbln4 were selective for NRXN variants containing splice segment (S) 4. Affinities for NRXNs estimated by surface plasmon resonance analysis were variable among Cbln subtypes. Cbln1 showed higher affinities to NRXNs than Cbln2, while the binding ability of Cbln4 was much lower than those of Cbln1 and Cbln2. The affinities of Cbln1 and Cbln2 were comparable between NRXN1α and NRXN1β, but those for NRXN2β and NRXN3β were lower. These results suggest that Cbln subtypes exert synaptogenic activities in corticalneurons by differentially interacting with NRXN variants containing S4.

In the murine brain, the first post-mitotic corticalneurons formed during embryogenesis express store-operated channels (SOCs) sensitive to Pyr3, initially proposed as a blocker of the transient receptor potential channel of C type 3 (TRPC3 channel). However, Pyr3 does not discriminate between Orai and TRPC3 channels, questioning the contribution of TRPC3 in SOCs. This study was undertaken to clarify the molecular identity and the pharmacological profile of native SOCs from E13 corticalneurons. The mRNA expression of STIM1-2 and Orai1-3 was assessed by quantitative reverse transcription polymerase chain reaction. E13 corticalneurons expressed STIM1-2 mRNAs, with STIM2 being the predominant isoform. Only transcripts of Orai2 were found but no Orai1 and Orai3 mRNAs. Blockers of Orai and TRPC channels (Pyr6, Pyr10, EVP4593, SAR7334, and GSK-7975A) were used to further characterize the endogenous SOCs. Their activity was recorded using the fluorescent Ca2+ probe Fluo-4. Cortical SOCs were sensitive to the Orai blockers Pyr6 and GSK-7975A, as well as to EVP4593, zinc, copper, and gadolinium ions, the latter one being the most potent SOCs blocker tested (IC50 ∼10 nM). SOCs were insensitive to the TRPC channel blockers Pyr10 and SAR7334. In addition, preventing mitochondrial Ca2+ uptake inhibited SOCs which were unaffected by inhibitors of the Ca2+-independent phospholipase A2. Altogether, Orai2 channels are present at the beginning of the embryonic murine cortico-genesis and form the core component of native SOCs in the immature cortex. This Ca2+ route is likely to play a role in the formation of the brain cortex. PMID:28018223

The aim of this study was to investigate the relationship between cell cycle reentry and apoptosis in cultured corticalneurons following oxygen-glucose deprivation (OGD). We found that the percentage of neurons with BrdU uptake, TUNEL staining, and colocalized BrdU uptake and TUNEL staining was increased relative to control 6, 12 and 24 h after 1 h of OGD. The number of neurons with colocalized BrdU and TUNEL staining was decreased relative to the number of TUNEL-positive neurons at 24 h. The expression of phosphorylated retinoblastoma protein (phospho-Rb) was significantly increased 6, 12 and 24 h after OGD, parallel with the changes in BrdU uptake. Phospho-Rb and TUNEL staining were colocalized in neurons 6 and 12 h after OGD. This colocalization was strikingly decreased 24 h after OGD. Treatment with the cyclin-dependent kinase inhibitor roscovitine (100 μM) decreased the expression of phospho-Rb and reduced neuronal apoptosis in vitro. These results demonstrated that attempted cell cycle reentry with phosphorylation of Rb induce early apoptosis in neurons after OGD and there must be other mechanisms involved in the later stages of neuronal apoptosis besides cell cycle reentry. Phosphoralated Rb may be an important factor which closely associates aberrant cell cycle reentry with the early stages of neuronal apoptosis following ischemia/hypoxia in vitro, and pharmacological interventions for neuroprotection may be useful directed at this keypoint.

Neurons firing spontaneously in bursts in the absence of synaptic transmission have been previously recorded in different layers of cortical brain slices. It has been suggested that such neurons could contribute to the generation of alternating UP and DOWN states, a pattern of activity seen during slow-wave sleep. Here, we show that in layer 6b (L6b), known from our previous studies to contain neurons highly responsive to the wake-promoting transmitter hypocretin/orexin (hcrt/orx), there is a set of neurons, endowed with distinct intrinsic properties, which displayed a strong propensity to fire spontaneously in rhythmic bursts. In response to small depolarizing steps, they responded with a delayed firing of action potentials which, upon higher depolarizing steps, invariably inactivated and were followed by a depolarized plateau potential and a depolarizing afterpotential. These cells also displayed a strong hyperpolarization-activated rectification compatible with the presence of an Ih current. Most L6b neurons with such properties were able to fire spontaneously in bursts. Their bursting activity was of intrinsic origin as it persisted not only in presence of blockers of ionotropic glutamatergic and GABAergic receptors but also in a condition of complete synaptic blockade. However, a small number of these neurons displayed a mix of intrinsic bursting and synaptically driven recurrent UP and DOWN states. Most of the bursting L6b neurons were depolarized and excited by hcrt/orx through a direct postsynaptic mechanism that led to tonic firing and eventually inactivation. Similarly, they were directly excited by noradrenaline, histamine, dopamine, and neurotensin. Finally, the intracellular injection of these cells with dye and their subsequent Neurolucida reconstruction indicated that they were spiny non-pyramidal neurons. These results lead us to suggest that the propensity for slow rhythmic bursting of this set of L6b neurons could be directly impeded by hcrt

Full Text Available BACKGROUND: Y2 receptor signalling is known to be important in neuropeptide Y (NPY-mediated effects on energy homeostasis and bone physiology. Y2 receptors are located post-synaptically as well as acting as auto receptors on NPY-expressing neurons, and the different roles of these two populations of Y2 receptors in the regulation of energy homeostasis and body composition are unclear. METHODOLOGY/PRINCIPAL FINDINGS: We thus generated two conditional knockout mouse models, Y2(lox/lox and NPYCre/+;Y2(lox/lox, in which Y2 receptors can be selectively ablated either in the hypothalamus or specifically in hypothalamic NPY-producing neurons of adult mice. Specific deletion of hypothalamic Y2 receptors increases food intake and body weight compared to controls. Importantly, specific ablation of hypothalamic Y2 receptors on NPY-containing neurons results in a significantly greater adiposity in female but not male mice, accompanied by increased hepatic triglyceride levels, decreased expression of liver carnitine palmitoyltransferase (CPT1 and increased expression of muscle phosphorylated acetyl-CoA carboxylase (ACC. While food intake, body weight, femur length, bone mineral content, density and cortical bone volume and thickness are not significantly altered, trabecular bone volume and number were significantly increased by hypothalamic Y2 deletion on NPY-expressing neurons. Interestingly, in situ hybridisation reveals increased NPY and decreased proopiomelanocortin (POMC mRNA expression in the arcuate nucleus of mice with hypothalamus-specific deletion of Y2 receptors in NPY neurons, consistent with a negative feedback mechanism between NPY expression and Y2 receptors on NPY-ergic neurons. CONCLUSIONS/SIGNIFICANCE: Taken together these data demonstrate the anti-obesogenic role of Y2 receptors in the brain, notably on NPY-ergic neurons, possibly via inhibition of NPY neurons and concomitant stimulation of POMC-expressing neurons in the arcuate nucleus of

Calcium imaging of the spontaneous activity in cortical slices has revealed repeating spatiotemporal patterns of transitions between so-called down states and up states (Ikegaya et al., 2004). Here we fit a model network of stochastic binary neurons to data from these experiments, and in doing so reproduce the distributions of such patterns. We use two versions of this model: (1) an unconnected network in which neurons are activated as independent Poisson processes; and (2) a network with an interaction matrix, estimated from the data, representing effective interactions between the neurons. The unconnected model (model 1) is sufficient to account for the statistics of repeating patterns in 11 of the 15 datasets studied. Model 2, with interactions between neurons, is required to account for pattern statistics of the remaining four. Three of these four datasets are the ones that contain the largest number of transitions, suggesting that long datasets are in general necessary to render interactions statistically visible. We then study the topology of the matrix of interactions estimated for these four datasets. For three of the four datasets, we find sparse matrices with long-tailed degree distributions and an overrepresentation of certain network motifs. The remaining dataset exhibits a strongly interconnected, spatially localized subgroup of neurons. In all cases, we find that interactions between neurons facilitate the generation of long patterns that do not repeat exactly.

In the present work we set out to investigate the neuroprotective effects of noscapine (0.5-2 µM) in presence of D-glucose on primary murine foetal corticalneurons after oxygen-glucose deprivation/24 h. recovery. Cell viability, nitric oxide production and intracellular calcium ((ca(2+))i) levels were evaluated by MTT assay, the modified Griess method and Fura-2 respectively. 25 and 100 mM D-glucose could, in a concentration dependent manner, improve cell viability and decrease NO production and (ca(2+))i level in neuronal cells after ischemic insult. Moreover, pre-incubation of cells with noscapine, noticeably enhanced protective effects of 25 and 100 mM D-glucose compared to similar conditions without noscapine pre-treatment. In fact, noscapine attenuated NO production in a dose-dependent fashion, after 30 minutes (min) OGD, during high-glucose (HG) condition in corticalneurons. Pretreatment with 2 μM noscapine and 25 or 100 mM D-glucose, was shown to decrease the rise in (ca(2+))i induced by Sodium azide/glucose deprivation (chemical OGD) model. These effects were more pronounced than that of 25 or 100 mM D-glucose alone. The present study demonstrated that the neuroprotective effects of HG before an ischemic insult were augmented by pre-treatment with noscapine. Our results also suggested that the neuroprotection offered by both HG and noscapine involve attenuation of NO production and (ca(2+))i levels stimulated by the experimental ischemia in corticalneurons.

Full Text Available The rodent stress hormone corticosterone changes neuronal activity in a slow and persistent manner through transcriptional regulation. In the rat dorsal hippocampus, corticosterone enhances the amplitude of calcium-dependent potassium currents that cause a lingering slow after-hyperpolarization (sAHP at the end of depolarizing events. In this study we compared the putative region-dependency of the delayed effects of corticosterone (approximately 5 hrs after treatment on sAHP as well as other active and passive properties of layer 2/3 pyramidal neurons from three prefrontal areas, i.e. the lateral orbitofrontal, prelimbic and infralimbic cortex, with the hippocampus of adult mice. In agreement with previous studies, corticosterone increased sAHP amplitude in the dorsal hippocampus with depolarizing steps of increasing amplitude. However, in the lateral orbitofrontal, prelimbic and infralimbic cortices we did not observe any modifications of sAHP amplitude after corticosterone treatment. Properties of single action potentials or % ratio of the last spike interval with respect to the first spike interval, an indicator of accommodation in an action potential train, were not significantly affected by corticosterone in all brain regions examined. Lastly, corticosterone treatment did not induce any lasting changes in passive membrane properties of hippocampal or corticalneurons. Overall, the data indicate that corticosterone slowly and very persistently increases the sAHP amplitude in hippocampal pyramidal neurons, while this is not the case in the cortical regions examined. This implies that changes in excitability across brain regions reached by corticosterone may vary over a prolonged period of time after stress.

Glucose is the major energy substrate in brain, however, during ketogenesis induced by starvation or prolonged hypoglycemia, the ketone bodies (KB), acetoacetate and β-hydroxybutyrate (BHB) can substitute for glucose. KB improve neuronal survival in diverse injury models, but the mechanisms by which KB prevent neuronal damage are still not well understood. In the present study we have investigated whether protection by the D isomer of BHB (D-BHB) against neuronal death induced by glucose deprivation (GD), is related to autophagy. Autophagy is a lysosomal-dependent degradation process activated during nutritional stress, which leads to the digestion of damaged proteins and organelles providing energy for cell survival. Results show that autophagy is activated in cortical cultured neurons during GD, as indicated by the increase in the levels of the lipidated form of the microtubule associated protein light chain 3 (LC3-II), and the number of autophagic vesicles. At early phases of glucose reintroduction (GR), the levels of p62 declined suggesting that the degradation of the autophagolysosomal content takes place at this time. In cultures exposed to GD and GR in the presence of D-BHB, the levels of LC3-II and p62 rapidly declined and remained low during GR, suggesting that the KB stimulates the autophagic flux preventing autophagosome accumulation and improving neuronal survival.

This paper describes the adhesion and growth of dissociated corticalneurons on chemically patterned surfaces over a time period of 30 days. The presence of neurons was demonstrated by measurement of spontaneous bioelectrical activity on a micropatterned multielectrode array. Chemical patterns were

A polymorphism of human AE3 is associated with idiopathic generalized epilepsy. Knockout of AE3 in mice lowers the threshold for triggering epileptic seizures. The explanations for these effects are elusive. Comparisons of cells from wild-type vs. AE3(-/-) mice show that AE3 (present in hippocampal neurons, not astrocytes; mediates HCO3(-) efflux) enhances intracellular pH (pHi ) recovery (decrease) from alkali loads in neurons and, surprisingly, adjacent astrocytes. During metabolic acidosis (MAc), AE3 speeds initial acidification, but limits the extent of pHi decrease in neurons and astrocytes. AE3 speeds re-alkalization after removal of MAc in neurons and astrocytes, and speeds neuronal pHi recovery from an ammonium prepulse-induced acid load. We propose that neuronal AE3 indirectly increases acid extrusion in (a) neurons via Cl(-) loading, and (b) astrocytes by somehow enhancing NBCe1 (major acid extruder). The latter would enhance depolarization-induced alkalinization of astrocytes, and extracellular acidification, and thereby reduce susceptibility to epileptic seizures. The anion exchanger AE3, expressed in hippocampal (HC) neurons but not astrocytes, contributes to intracellular pH (pHi ) regulation by facilitating the exchange of extracellular Cl(-) for intracellular HCO3(-) . The human AE3 polymorphism A867D is associated with idiopathic generalized epilepsy. Moreover, AE3 knockout (AE3(-/-) ) mice are more susceptible to epileptic seizure. The mechanism of these effects has been unclear because the starting pHi in AE3(-/-) and wild-type neurons is indistinguishable. The purpose of the present study was to use AE3(-/-) mice to investigate the role of AE3 in pHi homeostasis in HC neurons, co-cultured with astrocytes. We find that the presence of AE3 increases the acidification rate constant during pHi recovery from intracellular alkaline loads imposed by reducing [CO2 ]. The presence of AE3 also speeds intracellular acidification during the early phase of

The "dissociative " general anesthetic ketamine is a well-known N-methyl-D-aspartate receptor antagonist. However, whether ketamine, at clinically relevant concentrations, increases the activity of inhibitory γ-aminobutyric acid (GABA) receptor type A (GABAA) receptors in different brain regions remains controversial. Here, the authors studied the effects of ketamine on synaptic and extrasynaptic GABAA receptors in hippocampal neurons. Ketamine modulation of extrasynaptic GABAA receptors in corticalneurons was also examined. Whole cell currents were recorded from cultured murine neurons. Current evoked by exogenous GABA, miniature inhibitory postsynaptic currents, and currents directly activated by ketamine were studied. Ketamine did not alter the amplitude, frequency, or kinetics of postsynaptic currents but increased a tonic inhibitory current generated by extrasynaptic GABAA receptors in hippocampal neurons. For example, ketamine (100 µM) increased the tonic current by 33.6 ± 6.5% (mean ± SEM; 95% CI, 18.2 to 48.9; n = 8, P Ketamine shifted the GABA concentration-response curve to the left, but only when GABAA receptors were activated by low concentrations of GABA (n = 6). The selective increase in tonic current was attributed to ketamine increasing the apparent potency of GABA at high-affinity extrasynaptic GABAA receptors. Ketamine also increased a tonic current in corticalneurons (n = 11). Ketamine directly gated the opening of GABAA receptors, but only at high concentrations that are unlikely to occur during clinical use. Clinically relevant concentrations of ketamine increased the activity of high-affinity extrasynaptic GABAA receptors in the hippocampus and cortex, an effect that likely contributes to ketamine's neurodepressive properties.

Neurons in the rostral superior temporal sulcus (STS) are responsive to displays of body movements. We employed a parametric action space to determine how similarities among actions are represented by visual temporal neurons and how form and motion information contributes to their responses. The stimulus space consisted of a stick-plus-point-light figure performing arm actions and their blends. Multidimensional scaling showed that the responses of temporal neurons represented the ordinal similarity between these actions. Further tests distinguished neurons responding equally strongly to static presentations and to actions ("snapshot" neurons), from those responding much less strongly to static presentations, but responding well when motion was present ("motion" neurons). The "motion" neurons were predominantly found in the upper bank/fundus of the STS, and "snapshot" neurons in the lower bank of the STS and inferior temporal convexity. Most "motion" neurons showed strong response modulation during the course of an action, thus responding to action kinematics. "Motion" neurons displayed a greater average selectivity for these simple arm actions than did "snapshot" neurons. We suggest that the "motion" neurons code for visual kinematics, whereas the "snapshot" neurons code for form/posture, and that both can contribute to action recognition, in agreement with computation models of action recognition.

We report here the presence of fast subthreshold oscillatory potentials recorded in vitro from neurons within layer 4 of the guinea pig frontal cortex. Two types of oscillatory neurons were recorded: (i) One type exhibited subthreshold oscillations whose frequency increased with membrane depolarization and encompassed a range of 10-45 Hz. Action potentials in this type of neuron demonstrated clear after-hyperpolarizations. (ii) The second type of neuron was characterized by narrow-frequency oscillations near 35-50 Hz. These oscillations often outlasted the initiating depolarizing stimulus. No calcium component could be identified in their action potential. In both types of cell the subthreshold oscillations were tetrodotoxin-sensitive, indicating that the depolarizing phase of the oscillation was generated by a voltage-dependent sodium conductance. The initial depolarizing phase was followed by a potassium conductance responsible for the falling phase of the oscillatory wave. In both types of cell, the subthreshold oscillation could trigger spikes at the oscillatory frequency, if the membrane was sufficiently depolarized. Combining intracellular recordings with Lucifer yellow staining showed that the narrow-frequency oscillatory activity was produced by a sparsely spinous interneuron located in layer 4 of the cortex. This neuron has extensive local axonal collaterals that ramify in layers 3 and 4 such that they may contribute to the columnar synchronization of activity in the 40- to 50-Hz range. Cortical activity in this frequency range has been proposed as the basis for the "conjunctive properties" of central nervous system networks.

Nicotinamide mononucleotide adenylyl transferase 2 (NMNAT2) is a key neuronal maintenance factor and provides potent neuroprotection in numerous preclinical models of neurological disorders. NMNAT2 is significantly reduced in Alzheimer’s, Huntington’s, Parkinson’s diseases. Here we developed a Meso Scale Discovery (MSD)-based screening platform to quantify endogenous NMNAT2 in corticalneurons. The high sensitivity and large dynamic range of this NMNAT2-MSD platform allowed us to screen the Sigma LOPAC library consisting of 1280 compounds. This library had a 2.89% hit rate, with 24 NMNAT2 positive and 13 negative modulators identified. Western analysis was conducted to validate and determine the dose-dependency of identified modulators. Caffeine, one identified NMNAT2 positive-modulator, when systemically administered restored NMNAT2 expression in rTg4510 tauopathy mice to normal levels. We confirmed in a cell culture model that four selected positive-modulators exerted NMNAT2-specific neuroprotection against vincristine-induced cell death while four selected NMNAT2 negative modulators reduced neuronal viability in an NMNAT2-dependent manner. Many of the identified NMNAT2 positive modulators are predicted to increase cAMP concentration, suggesting that neuronal NMNAT2 levels are tightly regulated by cAMP signaling. Taken together, our findings indicate that the NMNAT2-MSD platform provides a sensitive phenotypic screen to detect NMNAT2 in neurons. PMID:28266613

The ability of organisms to categorize diverse and often novel stimuli depends on ongoing interactions with their environment. In a modality such as vision, categorization requires the generation of both selective and invariant responses of corticalneurons to complex visual stimuli. How does behavior contribute to shaping the responses of these neurons? Analysis of this question is made difficult by the complex multilevel interactions between many neural and behavioral variables. To mitigate this difficulty, we studied the development and ongoing plasticity of pattern-selective neuronal responses by means of synthetic neural modeling. For this purpose, we constructed Darwin V, which consists of a simulated neuronal model embedded in a real-world device that is capable of motion and autonomous behavior. The neuronal model consists of four major components: a visual system (containing cortical and subcortical networks); a taste system based on conductance; sets of motor neurons capable of triggering behavior; and a diffuse ascending (value) system. The modeled visual cortex consists of two areas: a topographic map responsive to elementary features connected to a higher-order map composed of initially non-selective neuronal units. During behavior over time in its environment, Darwin V encounters numerous objects consisting of black metal cubes displaying different patterns of white blobs and stripes. Initially, the lack of specific higher-order visual responses does not allow visual pattern discrimination, and appetitive and aversive behaviors are triggered by the 'taste' (surface conductivity of objects) alone. In the course of sensory experience, however, changes occur in visual and sensorimotor connection strengths, with two major consequences. First, units within the higher visual area acquire responses that are both pattern selective and translation invariant. Second, as a result of the operation of the value system, these responses become linked to appropriate

Full Text Available Abstract Background The secreted ligand Reelin is believed to regulate the translocation of prospective layer 6 (L6 neocortical neurons into the preplate, a loose layer of pioneer neurons that overlies the ventricular zone. Recent studies have also suggested that Reelin controls neuronal orientation and polarized dendritic growth during this period of early cortical development. To explicitly characterize and quantify how Reelin controls this critical aspect of neurite initiation and growth we used a new ex utero explant model of early cortical development to selectively label a subset of L6 corticalneurons for complete 3-D reconstruction. Results The total neurite arbor sizes of neurons in Reelin-deficient (reeler mutant and Dab1-deficient (Reelin-non-responsive scrambler mutant cortices were quantified and unexpectedly were not different than control arbor lengths (p = 0.51. For each mutant, however, arbor organization was markedly different: mutant neurons manifested more primary processes (neurites emitted directly from the soma than wild type, and these neurites were longer and displayed less branching. Reeler and scrambler mutant neurites extended tangentially rather than radially, and the Golgi apparatus that normally invests the apical neurite was compact in both reeler and scrambler mutants. Mutant cortices also exhibited a neurite “exclusion zone” which was relatively devoid of L6 neuron neurites and extended at least 15 μm beneath the pial surface, an area corresponding to the marginal zone (MZ in the wild type explants. The presence of an exclusion zone was also indicated in the orientation of mutant primary neurite and neuronal somata, which failed to adopt angles within ~20˚ of the radial line to the pial surface. Injection of recombinant Reelin to reeler, but not scrambler, mutant cortices fully rescued soma orientation, Golgi organization, and dendritic projection defects within four hrs. Conclusions These findings

Free radical-mediated oxidative damage to proteins, lipids, and DNA occurs in neurons during acute brain injuries and in neurodegenerative disorders. Membrane lipid peroxidation contributes to neuronal dysfunction and death, in part by disrupting neuronal ion homeostasis and cellular bioenergetics. Emerging findings suggest that 4-hydroxynonenal (HNE), an aldehyde produced during lipid peroxidation, impairs the function of various proteins involved in neuronal homeostasis. Here we tested the hypothesis that HNE impairs the cellular system that removes damaged proteins and organelles, the autophagy-lysosome pathway in rat primary corticalneurons. We found that HNE, at a concentration that causes apoptosis over a 48-72 h period, increases protein levels of LC3 II and p62 and within 1 and 4 h of exposure, respectively; LC3 II and p62 immunoreactive puncta were observed in the cytoplasm of HNE-treated neurons at 6 h. The extent of up-regulation of p62 and LC3 II in response to HNE was not affected by co-treatment with the lysosome inhibitor bafilomycin A1, suggesting that the effects of HNE on autophagy were secondary to lysosome inhibition. Indeed, we found that neurons exposed to HNE exhibit elevated pH levels, and decreased protein substrate hydrolysis and cathepsin B activity. Neurons exposed to HNE also exhibited the accumulation of K63-linked polyubiquitinated proteins, which are substrates targeted for lysosomal degradation. Moreover, we found that the levels of LAMP2a and constitutively active heat-shock protein 70, and numbers of LAMP2a-positive lysosomes, are decreased in neurons exposed to HNE. Our findings demonstrate that the lipid peroxidation product HNE causes early impairment of lysosomes which may contribute to the accumulation of damaged and dysfunctional proteins and organelles and consequent neuronal death. Because impaired lysosome function is increasingly recognized as an early event in the neuronal death that occurs in neurodegenerative

The primate cingulate gyrus contains multiple cortical areas that can be distinguished by several neurochemical features, including the distribution of neurofilament protein-enriched pyramidal neurons. In addition, connectivity and functional properties indicate that there are multiple motor areas in the cortex lining the cingulate sulcus. These motor areas were targeted for analysis of potential interactions among regional specialization, connectivity, and cellular characteristics such as neurochemical profile and morphology. Specifically, intracortical injections of retrogradely transported dyes and intracellular injection were combined with immunocytochemistry to investigate neurons projecting from the cingulate motor areas to the putative forelimb region of the primary motor cortex, area M1. Two separate groups of neurons projecting to area M1 emanated from the cingulate sulcus, one anterior and one posterior, both of which furnished commissural and ipsilateral connections with area M1. The primary difference between the two populations was laminar origin, with the anterior projection originating largely in deep layers, and the posterior projection taking origin equally in superficial and deep layers. With regard to cellular morphology, the anterior projection exhibited more morphologic diversity than the posterior projection. Commissural projections from both anterior and posterior fields originated largely in layer VI. Neurofilament protein distribution was a reliable tool for localizing the two projections and for discriminating between them. Comparable proportions of the two sets of projection neurons contained neurofilament protein, although the density and distribution of the total population of neurofilament protein-enriched neurons was very different in the two subareas of origin. Within a projection, the participating neurons exhibited a high degree of morphologic heterogeneity, and no correlation was observed between somatodendritic morphology and

The CB1 cannabinoid receptor, the main target of Δ(9)-tetrahydrocannabinol (THC), the most prominent psychoactive compound of marijuana, plays a crucial regulatory role in brain development as evidenced by the neurodevelopmental consequences of its manipulation in animal models. Likewise, recreational cannabis use during pregnancy affects brain structure and function of the progeny. However, the precise neurobiological substrates underlying the consequences of prenatal THC exposure remain unknown. As CB1 signaling is known to modulate long-range corticofugal connectivity, we analyzed the impact of THC exposure on cortical projection neuron development. THC administration to pregnant mice in a restricted time window interfered with subcerebral projection neuron generation, thereby altering corticospinal connectivity, and produced long-lasting alterations in the fine motor performance of the adult offspring. Consequences of THC exposure were reminiscent of those elicited by CB1 receptor genetic ablation, and CB1-null mice were resistant to THC-induced alterations. The identity of embryonic THC neuronal targets was determined by a Cre-mediated, lineage-specific, CB1 expression-rescue strategy in a CB1-null background. Early and selective CB1 reexpression in dorsal telencephalic glutamatergic neurons but not forebrain GABAergic neurons rescued the deficits in corticospinal motor neuron development of CB1-null mice and restored susceptibility to THC-induced motor alterations. In addition, THC administration induced an increase in seizure susceptibility that was mediated by its interference with CB1-dependent regulation of both glutamatergic and GABAergic neuron development. These findings demonstrate that prenatal exposure to THC has long-lasting deleterious consequences in the adult offspring solely mediated by its ability to disrupt the neurodevelopmental role of CB1 signaling.

Full Text Available Mental functions involve coordinated activities of specific neuronal ensembles that are embedded in complex brain circuits. Aberrant neuronal ensemble dynamics is thought to form the neurobiological basis of mental disorders. A major challenge in mental health research is to identify these cellular ensembles and determine what molecular mechanisms constrain their emergence and consolidation during development and learning. Here, we provide a perspective based on recent studies that use activity-dependent gene Arc/Arg3.1 as a cellular marker to identify neuronal ensembles and a molecular probe to modulate circuit functions. These studies have demonstrated that the transcription of Arc is activated in selective groups of frontal corticalneurons in response to specific behavioral tasks. Arc expression regulates the persistent firing of individual neurons and predicts the consolidation of neuronal ensembles during repeated learning. Therefore, the Arc pathway represents a prototypical example of activity-dependent genetic feedback regulation of neuronal ensembles. The activation of this pathway in the frontal cortex starts during early postnatal development and requires dopaminergic input. Conversely, genetic disruption of Arc leads to a hypoactive mesofrontal dopamine circuit and its related cognitive deficit. This mutual interaction suggests an auto-regulatory mechanism to amplify the impact of neuromodulators and activity-regulated genes during postnatal development. Such a mechanism may contribute to the association of mutations in dopamine and Arc pathways with neurodevelopmental psychiatric disorders. As the mesofrontal dopamine circuit shows extensive activity-dependent developmental plasticity, activity-guided modulation of dopaminergic projections or Arc ensembles during development may help to repair circuit deficits related to neuropsychiatric disorders.

Loss of the survival motor neuron gene (SMN1) is responsible for spinal muscular atrophy (SMA), the most common inherited cause of infant mortality. Even though the SMA phenotype is traditionally considered as related to spinal motor neuron loss, it remains debated whether the specific targeting of motor neurons could represent the best therapeutic option for the disease. We here investigated, using stereological quantification methods, the spinal cord and cerebral motor cortex of ∆7 SMA mice during development, to verify extent and selectivity of motor neuron loss. We found progressive post-natal loss of spinal motor neurons, already at pre-symptomatic stages, and a higher vulnerability of motor neurons innervating proximal and axial muscles. Larger motor neurons decreased in the course of disease, either for selective loss or specific developmental impairment. We also found a selective reduction of layer V pyramidal neurons associated with layer V gliosis in the cerebral motor cortex. Our data indicate that in the ∆7 SMA model SMN loss is critical for the spinal cord, particularly for specific motor neuron pools. Neuronal loss, however, is not selective for lower motor neurons. These data further suggest that SMA pathogenesis is likely more complex than previously anticipated. The better knowledge of SMA models might be instrumental in shaping better therapeutic options for affected patients.

How a mixture of acoustic sources is perceptually organized into discrete auditory objects remains unclear. One current hypothesis postulates that perceptual segregation of different sources is related to the spatiotemporal separation of cortical responses induced by each acoustic source or stream. In the present study, the dynamics of subthreshold membrane potential activity were measured across the entire tonotopic axis of the rodent primary auditory cortex during the auditory streaming paradigm using voltage-sensitive dye imaging. Consistent with the proposed hypothesis, we observed enhanced spatiotemporal segregation of cortical responses to alternating tone sequences as their frequency separation or presentation rate was increased, both manipulations known to promote stream segregation. However, across most streaming paradigm conditions tested, a substantial cortical region maintaining a response to both tones coexisted with more peripheral cortical regions responding more selectively to one of them. We propose that these coexisting subthreshold representation types could provide neural substrates to support the flexible switching between the integrated and segregated streaming percepts.

The dendritic spines on pyramidal cells represent the main postsynaptic elements of cortical excitatory synapses and they are fundamental structures in memory, learning and cognition. In the present study, we used intracellular injections of Lucifer yellow in fixed tissue to analyse over 19 500 dendritic spines that were completely reconstructed in three dimensions along the length of the basal dendrites of pyramidal neurons in the parahippocampal cortex and CA1 of patients with Alzheimer’s disease. Following intracellular injection, sections were immunostained for anti-Lucifer yellow and with tau monoclonal antibodies AT8 and PHF-1, which recognize tau phosphorylated at Ser202/Thr205 and at Ser396/404, respectively. We observed that the diffuse accumulation of phospho-tau in a putative pre-tangle state did not induce changes in the dendrites of pyramidal neurons, whereas the presence of tau aggregates forming intraneuronal neurofibrillary tangles was associated with progressive alteration of dendritic spines (loss of dendritic spines and changes in their morphology) and dendrite atrophy, depending on the degree of tangle development. Thus, the presence of phospho-tau in neurons does not necessarily mean that they suffer severe and irreversible effects as thought previously but rather, the characteristic cognitive impairment in Alzheimer’s disease is likely to depend on the relative number of neurons that have well developed tangles. PMID:23715095

Full Text Available Mitochondrial dysfunction plays a key role in the progression of Alzheimer's disease (AD. The accumulation of amyloid-beta peptide (Aβ in the brains of AD patients is thought to be closely related to neuronal mitochondrial dysfunction and oxidative stress. Therefore, protecting mitochondria from Aβ-induced neurotoxicity is an effective strategy for AD therapeutics. In a previous study, we found that geniposide, a pharmacologically active compound purified from gardenia fruit, has protective effects on oxidative stress and mitochondrial dysfunction in AD transgenic mouse models. However, whether geniposide has a protective effect on Aβ-induced neuronal dysfunction remains unknown. In the present study, we demonstrate that geniposide protects cultured primary corticalneurons from Aβ-mediated mitochondrial dysfunction by recovering ATP generation, mitochondrial membrane potential (MMP, and cytochrome c oxidase (CcO and caspase 3/9 activity; by reducing ROS production and cytochrome c leakage; as well as by inhibiting apoptosis. These findings suggest that geniposide may attenuate Aβ-induced neuronal injury by inhibiting mitochondrial dysfunction and oxidative stress.

Full Text Available Abstract Background Cortical development is a complex process that includes sequential generation of neuronal progenitors, which proliferate and migrate to form the stratified layers of the developing cortex. To identify the individual microRNAs (miRNAs and mRNAs that may regulate the genetic network guiding the earliest phase of cortical development, the expression profiles of rat neuronal progenitors obtained at embryonic day 11 (E11, E12 and E13 were analyzed. Results Neuronal progenitors were purified from telencephalic dissociates by a positive-selection strategy featuring surface labeling with tetanus-toxin and cholera-toxin followed by fluorescence-activated cell sorting. Microarray analyses revealed the fractions of miRNAs and mRNAs that were up-regulated or down-regulated in these neuronal progenitors at the beginning of cortical development. Nearly half of the dynamically expressed miRNAs were negatively correlated with the expression of their predicted target mRNAs. Conclusion These data support a regulatory role for miRNAs during the transition from neuronal progenitors into the earliest differentiating corticalneurons. In addition, by supplying a robust data set in which miRNA and mRNA profiles originate from the same purified cell type, this empirical study may facilitate the development of new algorithms to integrate various "-omics" data sets.

The present study examines the hypothesis that endogenous neural progenitor cells isolated from the neocortex of ischemic brain can differentiate into neurons or glial cells and contribute to neural regeneration. We performed middle cerebral artery occlusion to establish a model of cerebral ischemia/reperfusion injury in adult rats. Immunohistochemical staining of the cortex 1, 3, 7, 14 or 28 days after injury revealed that neural progenitor cells double-positive for nestin and sox-2 appeared in the injured cortex 1 and 3 days post-injury, and were also positive for glial ifbrillary acidic protein. New neurons were labeled using bromodeoxyuridine and different stages of maturity were identiifed using doublecortin, microtubule-associated protein 2 and neuronal nuclei antigen immunohistochemistry. Immature new neurons coexpressing doublecortin and bromodeoxyuridine were observed in the cortex at 3 and 7 days post-injury, and semi-mature and mature new neurons double-positive for microtubule-associated protein 2 and bromode-oxyuridine were found at 14 days post-injury. A few mature new neurons coexpressing neuronal nuclei antigen and bromodeoxyuridine were observed in the injured cortex 28 days post-injury. Glial ifbrillary acidic protein/bromodeoxyuridine double-positive astrocytes were also found in the injured cortex. Our ifndings suggest that neural progenitor cells are present in the damaged cortex of adult rats with cerebral ischemic brain injury, and that they differentiate into astrocytes and immature neurons, but most neurons fail to reach the mature stage.

This study examines the role of glucose and lactate as energy substrates to sustain synaptic vesicle cycling. Synaptic vesicle turnover was assessed in a quantitative manner by fluorescence microscopy in primary cultures of mouse corticalneurons. An electrode-equipped perfusion chamber was used to stimulate cells both by electrical field and potassium depolarization during image acquisition. An image analysis procedure was elaborated to select in an unbiased manner synaptic boutons loaded with the fluorescent dye N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl)pyridinium dibromide (FM1-43). Whereas a minority of the sites fully released their dye content following electrical stimulation, others needed subsequent K(+) depolarization to achieve full release. This functional heterogeneity was not significantly altered by the nature of metabolic substrates. Repetitive stimulation sequences of FM1-43 uptake and release were then performed in the absence of any metabolic substrate and showed that the number of active sites dramatically decreased after the first cycle of loading/unloading. The presence of 1 mM glucose or lactate was sufficient to sustain synaptic vesicle cycling under these conditions. Moreover, both substrates were equivalent for recovery of function after a phase of decreased metabolic substrate availability. Thus, lactate appears to be equivalent to glucose for sustaining synaptic vesicle turnover in cultured corticalneurons during activity.

In the cerebral cortex, projection neurons and interneurons work coordinately to establish neural networks for normal cortical functions. While the specific mechanisms that control productions of projection neurons and interneurons are beginning to be revealed, a global characterization of the molecular differences between these two neuron types is crucial for a more comprehensive understanding of their developmental specifications and functions. In this study, using lineage tracing power of combining Tbr2(Eomes)-GFP and Dcx-mRFP reporter mice, we prospectively separated intermediate progenitor cell (IPC)-derived neurons (IPNs) from non-IPC-derived neurons (non-IPNs) of the embryonic cerebral cortex. Molecular characterizations revealed that IPNs and non-IPNs were enriched with projection neurons and interneurons, respectively. Expression profiling documented cell-specific genes including differentially expressed transcriptional regulators that might be involved in cellular specifications, for instance, our data found that SOX1 and SOX2, which were known for important functions in neural stem/progenitor cells, continued to be expressed by interneurons but not by projection neurons. Transcriptome analyses of corticalneurons isolated at different stages of neurogenesis revealed distinct temporal patterns of expression of genes involved in early-born or late-born neuron specification. These data present a resource useful for further investigation of the molecular regulations and functions of projection neurons and interneurons.

AIM: To investigate the role of c-Jun N-terminal protein kinase 1 and 2 (JNK1/2) and the main signal pathway for its activation in hydrogen peroxide (H2O2) induced apoptotic-like cortical cell death. METHODS: Using the model of oxidative stress induced by H2O2, the expression and diphosphorylation of JNK1/2 was examined by immunoblotting analysis, and neuronal apoptotic like cell death was determined by 4',6-diamidino-2-phenylindole (DAPI) staining.RESULTS: The elevation in diphosphorylation level of JNK1/2 (4.40-/5.61-fold vs sham control) was associated with the concentration of H2O2 (0-100 μmol/L) and the development of apoptotic-like cell death (11.04 %-81.01%).There was no alteration of JNK1/2 protein expression following H2O2 treatment and recovery at different time points. Administration with JNK1/2 antisense oligonucleotides not only significantly decreased JNK1/2 protein expression and activation level, but also significantly reduced cortical cell death induced by H2O2 exposure.Furthermore, both JNK1/2 diphosphorylation and apoptotic-like cell death were largely prevented by pretreatment with (5S, l0R)-(-)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (MK-801)or omission of Ca2+ in incubation medium with ethylene glycol-bis(2-aminoethylether)-N,N,N',N-tetraacetic acid (EGTA). CONCLUSION: JNK1/2 is activated and participates in H2O2-induced apoptotic-like death in cultured rat corticalneurons mainly via N-methyl-D-aspartate (NMDA) receptor-mediated influx of extracellular Ca2+.

Full Text Available Ischemic stroke, characterized by the disturbance of the blood supply to the brain, is a severe worldwide health threat with high mortality and morbidity. However, there is no effective pharmacotherapy for ischemic injury. Currently, combined treatment is highly recommended for this devastating injury. In the present study, we investigated neuroprotective effects of the combination of omega-3 polyunsaturated fatty acids (ω-3 PUFAs and Lyciumbarbarum polysaccharide (LBP on corticalneurons using an in vitro ischemic model. Our study demonstrated that treatment with docosahexaenoic acid (DHA, a major component of the ω-3 PUFAs family, significantly inhibited the increase of intracellular Ca2+ in cultured wild type (WT corticalneurons subjected to oxygen-glucose deprivation/reperfusion (OGD/R injury and promoted their survival compared with the vehicle-treated control. The protective effects were further confirmed in cultured neurons with high endogenous ω-3 PUFAs that were isolated from fat-1 mice, in that a higher survival rate was found in fat-1 neurons compared with wild-type neurons after OGD/R injury. Our study also found that treatment with LBP (50 mg/L activated Trk-B signaling in corticalneurons and significantly attenuated OGD/R-induced cell apoptosis compared with the control. Notably, both combining LBP treatment with ω-3 PUFAs administration to WT neurons and adding LBP to fat-1 neurons showed enhanced effects on protecting corticalneurons against OGD/R injury via concurrently regulating the intracellular calcium overload and neurotrophic pathway. The results of the study suggest that ω-3 PUFAs and LBP are promising candidates for combined pharmacotherapy for ischemic stroke.

Ischemic stroke, characterized by the disturbance of the blood supply to the brain, is a severe worldwide health threat with high mortality and morbidity. However, there is no effective pharmacotherapy for ischemic injury. Currently, combined treatment is highly recommended for this devastating injury. In the present study, we investigated neuroprotective effects of the combination of omega-3 polyunsaturated fatty acids (ω-3 PUFAs) and Lyciumbarbarum polysaccharide (LBP) on corticalneurons using an in vitro ischemic model. Our study demonstrated that treatment with docosahexaenoic acid (DHA), a major component of the ω-3 PUFAs family, significantly inhibited the increase of intracellular Ca(2+) in cultured wild type (WT) corticalneurons subjected to oxygen-glucose deprivation/reperfusion (OGD/R) injury and promoted their survival compared with the vehicle-treated control. The protective effects were further confirmed in cultured neurons with high endogenous ω-3 PUFAs that were isolated from fat-1 mice, in that a higher survival rate was found in fat-1 neurons compared with wild-type neurons after OGD/R injury. Our study also found that treatment with LBP (50 mg/L) activated Trk-B signaling in corticalneurons and significantly attenuated OGD/R-induced cell apoptosis compared with the control. Notably, both combining LBP treatment with ω-3 PUFAs administration to WT neurons and adding LBP to fat-1 neurons showed enhanced effects on protecting corticalneurons against OGD/R injury via concurrently regulating the intracellular calcium overload and neurotrophic pathway. The results of the study suggest that ω-3 PUFAs and LBP are promising candidates for combined pharmacotherapy for ischemic stroke.

This study examines the role of c-jun N-terminal kinase (JNK) in mitochondrial signaling and bioenergetics in primary corticalneurons and isolated rat brain mitochondria. Exposure of neurons to either anisomycin (an activator of JNK/p38 mitogen-activated protein kinases) or H2O2 resulted in activation (phosphorylation) of JNK (mostly p46(JNK1)) and its translocation to mitochondria. Experiments with mitochondria isolated from either rat brain or primary corticalneurons and incubated with proteinase K revealed that phosphorylated JNK was associated with the outer mitochondrial membrane; this association resulted in the phosphorylation of the E(1alpha) subunit of pyruvate dehydrogenase, a key enzyme that catalyzes the oxidative decarboxylation of pyruvate and that links two major metabolic pathways: glycolysis and the tricarboxylic acid cycle. JNK-mediated phosphorylation of pyruvate dehydrogenase was not observed in experiments carried out with mitoplasts, thus suggesting the requirement of intact, functional mitochondria for this effect. JNK-mediated phosphorylation of pyruvate dehydrogenase was associated with a decline in its activity and, consequently, a shift to anaerobic pyruvate metabolism: the latter was confirmed by increased accumulation of lactic acid and decreased overall energy production (ATP levels). Pyruvate dehydrogenase appears to be a specific phosphorylation target for JNK, for other kinases, such as protein kinase A and protein kinase C did not elicit pyruvate dehydrogenase phosphorylation and did not decrease the activity of the complex. These results suggest that JNK mediates a signaling pathway that regulates metabolic functions in mitochondria as part of a network that coordinates cytosolic and mitochondrial processes relevant for cell function.

The establishment of a polarized morphology is essential for the development and function of neurons. During the development of the mammalian neocortex, neurons arise in the ventricular zone (VZ) from radial glia cells (RGCs) and leave the VZ to generate the cortical plate (CP). During their migration, newborn neurons first assume a multipolar morphology in the subventricular zone (SVZ) and lower intermediate zone (IZ). Subsequently, they undergo a multi-to-bipolar (MTB) transition to become bipolar in the upper IZ by developing a leading process and a trailing axon. The small GTPases Rap1A and Rap1B act as master regulators of neural cell polarity in the developing mouse neocortex. They are required for maintaining the polarity of RGCs and directing the MTB transition of multipolar neurons. Here we show that the Rap1 guanine nucleotide exchange factor (GEF) C3G (encoded by the Rapgef1 gene) is a crucial regulator of the MTB transition in vivo by conditionally inactivating the Rapgef1 gene in the developing mouse cortex at different time points during neuronal development. Inactivation of C3G results in defects in neuronal migration, axon formation and cortical lamination. Live cell imaging shows that C3G is required in corticalneurons for both the specification of an axon and the initiation of radial migration by forming a leading process.

Orthograde and retrograde tracers were used to examine subcortical connections of neurons in the neurological mutant tish rat. This animal exhibits bilateral heterotopia similar to those observed in epileptic humans with subcortical band heterotopia. Terminal varicosities were labeled in the striatum, thalamus, brainstem, and spinal cord following injections of the anterograde tracer biotinylated dextran amine (BDA) into the heterotopic cortex. The general topography of corticothalamic projections was evaluated by injecting the retrograde tracer Fluoro-Gold (FG) into ventral thalamic nuclei. Retrograde labeling of small-to-medium sized neurons was observed in layer VI of topographically restricted portions of the normotopic cortex. Similar appearing cells were labeled in the neighboring portions of the underlying heterotopia; however, these neurons did not display characteristic lamination or radial orientation. Thalamocortical terminals labeled by injecting BDA into the ventroposterolateral nucleus (VPL) were observed primarily in layer IV of the medial aspect of the normotopic somatosensory cortex. In contrast, a radial column of terminals was present in the underlying heterotopia. Typical barrel labeling was found in the lateral aspect of the normotopic somatosensory cortex after injecting the ventroposteromedial nucleus (VPM), whereas more diffuse patches of labeling were observed in the underlying heterotopia. Heterotopic neurons in the tish cortex, thus, exhibit characteristic features of subcortical connectivity. Both normotopic and heterotopic neurons in the tish brain project to appropriate subcortical sites and establish bidirectional topographic connections with the thalamus. These results suggest that primary sensory-motor information is represented in a parallel manner in the normotopic and heterotopic cortices of the tish rat.

How a mixture of acoustic sources is perceptually organized into discrete auditory objects remains unclear. One current hypothesis postulates that perceptual segregation of different sources is related to the spatiotemporal separation of cortical responses induced by each acoustic source or stream. In the present study, the dynamics of subthreshold membrane potential activity were measured across the entire tonotopic axis of the rodent primary auditory cortex during the auditory streaming paradigm using voltage-sensitive dye imaging. Consistent with the proposed hypothesis, we observed enhanced spatiotemporal segregation of cortical responses to alternating tone sequences as their frequency separation or presentation rate was increased, both manipulations known to promote stream segregation. However, across most streaming paradigm conditions tested, a substantial cortical region maintaining a response to both tones coexisted with more peripheral cortical regions responding more selectively to one of them. We propose that these coexisting subthreshold representation types could provide neural substrates to support the flexible switching between the integrated and segregated streaming percepts. PMID:26269558

Several seconds of adaptation to a flickered stimulus causes a subsequent brief static stimulus to appear longer in duration. Nonsensory factors, such as increased arousal and attention, have been thought to mediate this flicker-based temporal-dilation aftereffect. In this study, we provide evidence that adaptation of low-level cortical visual…

Full Text Available Interleukin-6 has been shown to be involved in nerve injury and nerve regeneration, but the effects of long-term administration of high concentrations of interleukin-6 on neurons in the central nervous system is poorly understood. This study investigated the effects of 24 hour exposure of interleukin-6 on corticalneurons at various concentrations (0.1, 1, 5 and 10 ng/mL and the effects of 10 ng/mL interleukin-6 exposure to corticalneurons for various durations (2, 4, 8, 24 and 48 hours by studying voltage-gated Na + channels using a patch-clamp technique. Voltage-clamp recording results demonstrated that interleukin-6 suppressed Na + currents through its receptor in a time- and dose-dependent manner, but did not alter voltage-dependent activation and inactivation. Current-clamp recording results were consistent with voltage-clamp recording results. Interleukin-6 reduced the action potential amplitude of corticalneurons, but did not change the action potential threshold. The regulation of voltage-gated Na + channels in rat corticalneurons by interleukin-6 is time- and dose-dependent.

Interleukin-6 has been shown to be involved in nerve injury and nerve regeneration, but the effects of long-term administration of high concentrations of interleukin-6 on neurons in the central nervous system is poorly understood. This study investigated the effects of 24 hour exposure of interleukin-6 on corticalneurons at various concentrations (0.1, 1, 5 and 10 ng/mL) and the effects of 10 ng/mL interleukin-6 exposure to corticalneurons for various durations (2, 4, 8, 24 and 48 hours) by studying voltage-gated Na(+) channels using a patch-clamp technique. Voltage-clamp recording results demonstrated that interleukin-6 suppressed Na(+) currents through its receptor in a time- and dose-dependent manner, but did not alter voltage-dependent activation and inactivation. Current-clamp recording results were consistent with voltage-clamp recording results. Interleukin-6 reduced the action potential amplitude of corticalneurons, but did not change the action potential threshold. The regulation of voltage-gated Na(+) channels in rat corticalneurons by interleukin-6 is time- and dose-dependent.

Interleukin-6 has been shown to be involved in nerve injury and nerve regeneration, but the effects of long-term administration of high concentrations of interleukin-6 on neurons in the central nervous system is poorly understood. This study investigated the effects of 24 hour expo-sure of interleukin-6 on corticalneurons at various concentrations (0.1, 1, 5 and 10 ng/mL) and the effects of 10 ng/mL interleukin-6 exposure to corticalneurons for various durations (2, 4, 8, 24 and 48 hours) by studying voltage-gated Na+ channels using a patch-clamp technique. Volt-age-clamp recording results demonstrated that interleukin-6 suppressed Na+ currents through its receptor in a time- and dose-dependent manner, but did not alter voltage-dependent activation and inactivation. Current-clamp recording results were consistent with voltage-clamp recording results. Interleukin-6 reduced the action potential amplitude of corticalneurons, but did not change the action potential threshold. The regulation of voltage-gated Na+channels in rat corti-calneurons by interleukin-6 is time- and dose-dependent.

We present a methodology for detecting effective connections between simultaneously recorded neurons using an information transmission measure to identify the presence and direction of information flow from one neuron to another. Using simulated and experimentally-measured data, we evaluate the performance of our proposed method and compare it to the traditional transfer entropy approach. In simulations, our measure of information transmission outperforms transfer entropy in identifying the effective connectivity structure of a neuron ensemble. For experimentally recorded data, where ground truth is unavailable, the proposed method also yields a more plausible effective connectivity structure than transfer entropy.

Accumulating evidence suggests that reducing neurite outgrowth and synaptic plasticity plays a critical role in the pathology of cognitive deficits in schizophrenia. The N-methyl-D-aspartate receptor antagonist phencyclidine (PCP) can induce symptoms of schizophrenia as well as reduce dendritic spine density and neurite growth. The antipsychotic drug olanzapine may improve these deficits. This study aimed to investigate: (1) if olanzapine prevents PCP-induced suppression of neurite outgrowth and synaptic protein expression; (2) if olanzapine affects the Akt-GSK3 signaling pathway; and (3) the role of neuregulin 1 (NRG1) in this process. Immunofluorescence revealed that PCP treatment for 24 hours reduces both neurite length (28.5%) and the number of neurite branches (35.6%) in primary prefrontal corticalneuron cultures. PCP reduced protein and mRNA expressions of synaptophysin (24.9% and 23.2%, respectively) and PSD95 (31.5% and 21.4%, respectively), and the protein expression of p-Akt (26.7%) and p-GSK3β (35.2%). Olanzapine co-treatment prevented these PCP-induced effects in normal neurons but not in neurons from NRG1-knockout mice. These results indicate that NRG1 mediates the preventive effects of olanzapine on the PCP-induced impairment of neurite outgrowth and synaptic protein expression. This study provides potential targets for interventions on improving the efficacy of olanzapine on preventing cognitive deficits in schizophrenia.

To explore the roles of astrocytes in the epileptogenesis, astrocytes and neurons were isolated, purified and cultured in vitro from cerebral cortex of rats. The astrocytes were activated by ciliary neurotrophic factor (CNTF) and astrocytic conditioned medium (ACM) was collected to treat neurons for 4, 8 and 12 h. By using Western blot, the expression of calmodulin dependent protein kinase II (CaMK II), inducible nitric oxide synthase (iNOS) and adenylate cyclase (AC) was detected in neurons. The results showed that the expression of CaMK II, iNOS and AC was increased significantly in the neurons treated with ACM from 4 h to 12 h (PACM and such signal pathways as NOS-NO-cGMP, Ca2+/CaM-CaMK II and AC-cAMP-PKA might take part in the signal transduction of epileptogenesis.

We present a methodology for detecting effective connections between simultaneously recorded neurons using an information transmission measure to identify the presence and direction of information flow from one neuron to another. Using simulated and experimentally-measured data, we evaluate the performance of our proposed method and compare it to the traditional transfer entropy approach. In simulations, our measure of information transmission outperforms transfer entropy in identifying the e...

Full Text Available Trade-off between reproducibility of neuronal activities and computational efficiency is one ofcrucial subjects in computational neuroscience and neuromorphic engineering. A wide variety ofneuronal models have been studied from different viewpoints. The digital spiking silicon neuron(DSSN model is a qualitative model that focuses on efficient implementation by digital arithmeticcircuits. We expanded the DSSN model and found appropriate parameter sets with which itreproduces the dynamical behaviors of the ionic-conductance models of four classes of corticaland thalamic neurons. We first developed a 4-variable model by reducing the number of variablesin the ionic-conductance models and elucidated its mathematical structures using bifurcationanalysis. Then, expanded DSSN models were constructed that reproduce these mathematicalstructures and capture the characteristic behavior of each neuron class. We confirmed thatstatistics of the neuronal spike sequences are similar in the DSSN and the ionic-conductancemodels. Computational cost of the DSSN model is larger than that of the recent sophisticatedIntegrate-and-Fire-based models, but smaller than the ionic-conductance models. This modelis intended to provide another meeting point for above trade-off that satisfies the demand forlarge-scale neuronal network simulation with closer-to-biology models.

It has been widely accepted that microglia, which are the innate immune cells in the brain, upon activation can cause neuronal damage. In the present study, we investigated the role of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in regulating microglial nitric oxide production and its role in causing neuronal damage. The study revealed that TCDD stimulates the expression of inducible nitric oxide synthase (iNOS) as well as the production of nitric oxide (NO) in a dose- and time-dependent manner. Further, a rapid activation of p38 and JNK MAPKs was found in HAPI microglia following TCDD treatment. Blockage of p38 and JNK kinases with their specific inhibitors, SB202190 and SP600125, significantly reduced TCDD-induced iNOS expression and NO production. In addition, it was demonstrated through treating rat primary corticalneurons with media conditioned with TCDD treated microglia that microglial iNOS activation mediates neuronal apoptosis. Lastly, it was also found that p38 and JNK MAPK inhibitors could attenuate the apoptosis of rat corticalneurons upon exposure to medium conditioned by TCDD-treated HAPI microglial cells. Based on these observations, we highlight that the p38/JNK MAPK pathways play an important role in TCDD-induced iNOS activation in rat HAPI microglia and in the subsequent induction of apoptosis in primary corticalneurons.

Full Text Available The mammalian brain consists of numerous compartments that are closely connected with each other via neural networks, comprising the basis of higher order brain functions. The highly specialized structure originates from simple pseudostratified neuroepithelium-derived neural progenitors located near the ventricle. A long journey by neurons from the ventricular side is essential for the formation of a sophisticated brain structure, including a mammalian-specific six-layered cerebral cortex. Neuronal migration consists of several contiguous steps, but the locomotion mode comprises a large part of the migration. The locomoting neurons exhibit unique features; a radial glial fiber-dependent migration requiring the endocytic recycling of N-cadherin and a neuron-specific migration mode with dilation/swelling formation that requires the actin and microtubule organization possibly regulated by cyclin-dependent kinase 5 (Cdk5, Dcx, p27kip1, Rac1 and POSH. Here I will introduce the roles of various cellular events, such as cytoskeletal organization, cell adhesion and membrane trafficking, in the regulation of the neuronal migration, with particular focus on the locomotion mode.

Physiological data suggest that in the CA1-CA3 hippocampal areas of rats, entorhinal cortical efferents directly influence the activity of interneurons, in addition to pyramidal cells. To verify this hypothesis, the following experiments were performed: 1) light microscopic double-immunostaining for parvalbumin and the anterograde tracer Phaseolus vulgaris-leucoagglutinin injected into the entorhinal cortex; 2) light and electron microscopic analysis of cleaved spectrin-immunostained (i.e., degenerating axons and boutons) hippocampal sections following entorhinal cortex lesion; and 3) an electron microscopic study of parvalbumin-immunostained hippocampal sections after entorhinal cortex lesion. The results demonstrate that in the stratum lacunosum-moleculare of the CA1 and CA3 regions, entorhinal cortical axons form asymmetric synaptic contacts on parvalbumin-containing dendritic shafts. In the stratum lacunosum-moleculare, parvalbumin-immunoreactive dendrites represent processes of GABAergic, inhibitory basket and chandelier cells; these interneurons innervate the perisomatic area and axon initial segments of pyramidal cells, respectively. A feed-forward activation of these neurons by the entorhinal input may explain the strong, short-latency inhibition of pyramidal cells.

Pyramidal cells of the cerebral cortex are born in the ventricular zone and migrate through the intermediate zone to enter into the cortical plate. In the intermediate zone, these migrating precursors move tangentially and initiate the extension of their axons by transiently adopting a characteristic multipolar morphology. We observe that expression of the forkhead transcription factor FoxG1 is dynamically regulated during this transitional period. By utilizing conditional genetic strategies, we show that the downregulation of FoxG1 at the beginning of the multipolar cell phase induces Unc5D expression, the timing of which ultimately determines the laminar identity of pyramidal neurons. In addition, we demonstrate that the re-expression of FoxG1 is required for cells to transit out of the multipolar cell phase and to enter into the cortical plate. Thus, the dynamic expression of FoxG1 during migration within the intermediate zone is essential for the proper assembly of the cerebral cortex.

Secretory and cytosolic phospholipases A2 (sPLA2 and cPLA2) may contribute to the release of arachidonic acid and other bioactive lipids, which are modulators of synaptic function. In primary corticalneuron cultures, neurotoxic cell death and [3H]arachidonate metabolism was studied after adding ...

Glutamate receptor (GluR) δ1 is widely expressed in the developing forebrain, whereas GluRδ2 is selectively expressed in cerebellar Purkinje cells. Recently, we found that trans-synaptic interaction of postsynaptic GluRδ2 and pre-synaptic neurexins (NRXNs) through cerebellin precursor protein (Cbln) 1 mediates excitatory synapse formation in the cerebellum. Thus, a question arises whether GluRδ1 regulates synapse formation in the forebrain. In this study, we showed that the N-terminal domain of GluRδ1 induced inhibitory presynaptic differentiation of some populations of cultured corticalneurons. When Cbln1 or Cbln2 was added to cultures, GluRδ1 expressed in HEK293T cells induced preferentially inhibitory presynaptic differentiation of cultured corticalneurons. The synaptogenic activity of GluRδ1 was suppressed by the addition of the extracellular domain of NRXN1α or NRXN1β containing splice segment 4. Cbln subtypes directly bound to the N-terminal domain of GluRδ1. The synaptogenic activity of GluRδ1 in the presence of Cbln subtypes correlated well with their binding affinities. When transfected to corticalneurons, GluRδ1 stimulated inhibitory synapse formation in the presence of Cbln1 or Cbln2. These results together with differential interactions of Cbln subtypes with NRXN variants suggest that GluRδ1 induces preferentially inhibitory presynaptic differentiation of corticalneurons by interacting with NRXNs containing splice segment 4 through Cbln subtypes.

We previously demonstrated that the P2X7 receptor (P2X7R), a purinergic receptor, expressed by mouse cultured cortical astrocytes is constitutively activated without any exogenous stimulus, differing from the case of neurons. It is well known that astrocytic morphology differs between in vitro and in vivo situations, implying different functionalities. Brain acute slices are widely accepted as an in vitro experimental system that reflects in vivo cell conditions better than in vitro cell culture ones. We examined whether astrocytic P2X7Rs exhibited constitutive activation in mouse cortical slices. In acute cortical slices, P2X7R-immunoreactivity was detected in both glial fibrillary acidic protein-immunopositive astrocytes and microtubule-associated protein 2-immunopositive neurons. Astrocytic, but not neuronal, spontaneous uptake of propidium iodide, an indicator of P2X7R channel/pore activity, was inhibited by representative antagonists of P2X7R, but they had no effect on the uptake by astrocytes in membrane-permeabilized fixed slices. These findings indicate that astrocytes, but not neurons, in acute cortical slices exhibit constitutive activation of P2X7Rs under non-stimulated resting conditions as in the case of cell culture systems.

Objective To explore the changes of protein expression of mito-fission gene dynaminrelated 1 (Drp 1) in the corticalneurons of rats with chronic fluorosis.MethodsA total of 120 one-month-old SD rats (each weighing approximately 100—120 g at the beginning of the

Postnatal development of physiological properties underlying slow intrathalamic oscillations was studied by whole-cell recording from synaptically coupled neurons of the reticular nucleus (RTN) and ventral posterior nucleus (VPN) of mouse brain slices in vitro and compared with the morphological development of dye-injected cells. Between postnatal days 3 and 11 (P3-P11), progressive changes in RTN and VPN neurons included shortening of the membrane time constant, decreasing input resistance, and lowering of the resting membrane potential (RMP). Low-threshold Ca2+ spikes (LTS) were present from P3, but their capacity to sustain multispike bursts was limited before P11. Synaptic responses were evoked in RTN and VPN neurons by electrical stimulation of the internal capsule from P3. Younger RTN neurons responded with a single spike, but their capacity to fire bursts gradually improved as the RMP reached levels below the LTS activation potential. Concomitantly, as the reversal potential of the inhibitory postsynaptic potential in VPN neurons became more negative, its capacity to deinactivate the LTS increased, and rebound bursts that could maintain oscillations were produced; sustained oscillations became the typical response to internal capsule stimulation at P12. The functional maturation of the intrathalamic circuitry, particularly between P10 and P14, occurs in parallel with the morphological maturation (size, dendritic growth, and dendritic field structure) of individual RTN and VPN neurons, as studied by confocal microscopy. Maturation of RTN cells led that of VPN cells by 2-3 d. The appearance of intrathalamic oscillations is probably correlated with the appearance of slow-wave sleep in postnatal animals.

Although the neural locus of strabismic amblyopia has been shown to lie at the first site of binocular integration, first in cat and then in primate, an adequate mechanism is still lacking. Here we hypothesise that increased temporal dispersion of LGN X-cell afferents driven by the deviating eye onto single corticalneurons may provide a neural mechanism for strabismic amblyopia. This idea was investigated via single cell extracellular recordings of 93 X and 50 Y type LGN neurons from strabismic and normal cats. Both X and Y neurons driven by the non-deviating eye showed shorter latencies than those driven by either the strabismic or normal eyes. Also the mean latency difference between X and Y neurons was much greater for the strabismic cells compared with the other two groups. The incidence of lagged X-cells driven by the deviating eye of the strabismic cats was higher than that of LGN X-cells from normal animals. Remarkably, none of the cells recorded from the laminae driven by the non-deviating eye were of the lagged class. A simple computational model was constructed in which a mixture of lagged and non-lagged afferents converge on to single corticalneurons. Model cut-off spatial frequencies to a moving grating stimulus were sensitive to the temporal dispersion of the geniculate afferents. Thus strabismic amblyopia could be viewed as a lack of developmental tuning of geniculate lags for neurons driven by the amblyopic eye. Monocular control of fixation by the non-deviating eye is associated with reduced incidence of lagged neurons, suggesting that in normal vision, lagged neurons might play a role in maintaining binocular connections for corticalneurons.

Background Cerebral ischemia-reperfusion injury is the main reason for the loss of neurons in the ischemic cerebrovascular disease. Therefore, to deeply understand its pathogenesis and find a new target is the key issue to be solved. This research aimed to investigate the neuroprotective effects of salvianolic acid B (SalB) against oxygen-glucose deprivation/reperfusion (OGD/RP) damage in primary rat corticalneurons.Methods The primary cultures of neonatal Wister rats were randomly divided into the control group, the OGD/RP group and the SalB-treatment group (10 mg/L). The cell model was established by depriving of oxygen and glucose for 3 hours and reperfusion for 3 hours and 24 hours, respectively. The neuron viability was determined by MTT assay. The level of cellular reactive oxygen species (ROS) was detected by fluorescent labeling method and spin trapping technique respectively. The activities of neuronal Mn-superoxide dismutase (Mn-SOD), catalase (CAT) and glutathione peroxidase (GSH-PX) were assayed by chromatometry. The mitochondria membrane potential (△ψm) was quantitatively analyzed by flow cytometry. The release rate of cytochrome c was detected by Western blotting. The neuronal ultrastructure was observed by transmission electron microscopy. Statistical significance was evaluated by analysis of variance (ANOVA)followed by Student-Newman-Keuls test.Results OGD/RP increased the level of cellular ROS, but decreased the cell viability and the activities of Mn-SOD, CAT and GSH-PX; SalB treatment significantly reduced the level of ROS (P ＜0.05); and enhanced the cell viability (P ＜0.05)and the activities of these antioxidases (P ＜0.05). Additionally, OGD/RP induced the fluorescence value of △ψm to diminish and the release rate of cytochrome c to rise notably; SalB markedly elevated the level of △ψm (P ＜0.01) and depressed the release rate of cytochrome c (P ＜0.05); it also ameliorated the neuronal morphological injury.Conclusion The

Retrograde labeling of neuronal elements in the brain and spinal cord has been investigated by autoradiographic techniques following injections of D-(/sup 3/H)aspartate (asp), (/sup 3/H)..gamma..-aminobutyric acid (GABA) or horseradish peroxidase (HRP) in the medulla and spinal cord of rats. Twenty-four hours after D-(/sup 3/H)asp injections focused upon the cuneate nucleus, autoradiographic labeling is present over fibers in the pyramidal tract, internal capsule and over layer V pyramids in the forelimb representation of the sensorimotor cortex. After (/sup 3/H)GABA injections in the same nucleus no labeling attributable to retrograde translocation can be detected in spinal segments, brain stem or cortex. Conversely, injections of 30% HRP in the cuneate nucleus label neurons in several brain stem nuclei, in spinal gray and in layer V of the sensorimotor cortex. D-(/sup 3/H)Asp injections focused on the dorsal horn at cervical segments label a fraction of perikarya of the substantia gelatinosa and a sparser population of larger neurons in laminae IV to VI for a distance of 3-5 segments above and below the injection point. No brain stem neuronal perikarya appear labeled following spinal injections of D-(/sup 3/H)asp although autoradiographic grains overlie pyramidal tract fibers on the side contralateral to the injection.

A detrimental effect of hyperglycemia in ischemic brain has been demonstrated in laboratory experiments and it has been found that hyperglycemia in ischemic stroke is a predictor of poor outcome. We determined serum neuron specific enolase (NSE) concentrations in 41 consecutive patients with a cereb

Advanced glycation end products lead to cell apoptosis, and cause cell death by increasing endoplasmic reticulum stress. Advanced glycation end products alone may also directly cause damage to tissues and cells, but the precise mechanism remains unknown. This study used primary cultures of rat cerebral cortex neurons, and treated cells with different concentrations of glycation end products (50, 100, 200, 400 mg/L), and with an antibody for the receptor of advanced glycation end products before and after treatment with advanced glycation end products. The results showed that with increasing concentrations of glycation end products, free radical content increased in neurons, and the number of apoptotic cells increased in a dose-dependent manner. Before and after treatment of advanced glycation end products, the addition of the antibody against advanced glycation end-products markedly reduced hydroxyl free radicals, malondialdehyde levels, and inhibited cell apoptosis. This result indicated that the antibody for receptor of advanced glycation end-products in neurons from the rat cerebral cortex can reduce glycation end product-induced oxidative stress damage by suppressing glycation end product receptors. Overall, our study confirms that the advanced glycation end products-advanced glycation end products receptor pathway may be the main signaling pathway leading to neuronal damage.

During systems consolidation, memories are spontaneously replayed favoring information transfer from hippocampus to neocortex. However, at present no empirically supported mechanism to accomplish a transfer of memory from hippocampal to extra-hippocampal sites has been offered. We used cultured neuronal networks on multielectrode arrays and…

This study is to investigate the effect of the effective components group of Xiaoshuantongluo (XECG) on neuronal injury induced by oxygen-glucose deprivation (OGD) in primary cortical cultures isolated from SD rat cortex at day 3 and the possible mechanism. Cells were divided into control group, OGD model group and XECG group (1, 3 and 10 mg x L(-1)). The cell viability was assessed with MTT assay and the LDH release rate was measured by enzyme label kit. The cell apoptosis was analyzed using Hoechst staining. RT-PCR was applied to detect the mRNA levels of JAK2 and STAT3. Western blotting was used to detect the expressions of Bcl-2, Bax, p-JAK2 and p-STAT3 proteins. Results showed that XECG resulted in an obvious resistance to oxygen-glucose deprivation-induced cell apoptosis and decrement of cell viability, decrease the cell LDH release rate. XECG could adjust the expression of Bcl-2 and Bax proteins and increase Bcl-2/Bax ratio, up-regulate the expression of p-JAK2 and p-STAT3. In conclusion, XECG could protect against the neuronal injury cells exposed to OGD, which may be relevant to the promotion of JAK2/STAT3 signaling pathway, and impact the expression of Bax and Bcl-2.

Full Text Available The neurofibrillary tau pathology and amyloid deposits seen in Alzheimer's disease (AD also have been seen in bacteria-infected brains. However, few studies have examined the role of these bacteria in the generation of tau pathology. One suggested link between infection and Alzheimer’s disease is edentulism, the complete loss of teeth. Edentulism can result from chronic periodontal disease due to infection by Enterococcus faecalis. The current study assessed the ability to generate early Alzheimer-like neurofibrillary epitopes in primary rat corticalneurons through bacterial infection by Enterococcus faecalis. Seven-day old cultured neurons were infected with Enterococcus faecalis for 24- and 48-hours. An upward molecular weight shift in tau by western blotting and increased appearance of tau reactivity in cell bodies and degenerating neurites was found in the 48-hour infection group for the antibody CP13 (phospho-Serine-202. A substantial increase in reactivity of Alz-50 was seen at 24- and 48- hours after infection. Furthermore, extensive MAP2 reactivity also was seen at 24- and 48-hours post-infection. Our preliminary findings suggest a potential link between Enterococcus faecalis infection and intracellular changes that may help facilitate early AD-like neurofibrillary pathology.

The neurofibrillary tau pathology and amyloid deposits seen in Alzheimer's disease (AD) also have been seen in bacteria-infected brains. However, few studies have examined the role of these bacteria in the generation of tau pathology. One suggested link between infection and AD is edentulism, the complete loss of teeth. Edentulism can result from chronic periodontal disease due to infection by Enterococcus faecalis. The current study assessed the ability to generate early Alzheimer-like neurofibrillary epitopes in primary rat corticalneurons through bacterial infection by E. faecalis. Seven-day old cultured neurons were infected with E. faecalis for 24 and 48 h. An upward molecular weight shift in tau by Western blotting (WB) and increased appearance of tau reactivity in cell bodies and degenerating neurites was found in the 48 h infection group for the antibody CP13 (phospho-Serine 202). A substantial increase in reactivity of Alz-50 was seen at 24 and 48 h after infection. Furthermore, extensive microtubule-associated protein 2 (MAP2) reactivity also was seen at 24 and 48 h post-infection. Our preliminary findings suggest a potential link between E. faecalis infection and intracellular changes that may help facilitate early AD-like neurofibrillary pathology. HighlightsEnterococcus faecalis used in the generation of AD neurofibrillary epitopes in rat.Infection increases Alz-50, phospho-Serine 202 tau, and MAP2 expression.Infection by Enterococcus may play a role in early Alzheimer neurofibrillary changes.

Aim: To investigate the mechanism of the action of estrogen, which stimulates the release of secreted amyloid precursor protein α (sAPPα) and decreases the gen eration of amyloid-β protein (Aβ), a dominant component in senile plaques in the brains of Alzheimer's disease patients. Methods: Experiments were carried out inprimary rat corticalneurons, and Western blot was used to detect sAPPα in aculture medium and the total amount of cellular amyloid precursor protein (APP) in neurons. Results: 17β-Estradiol (but not 17α-estradiol) and β-estradiol 6-(Ocarboxymethyl) oxime: BSA increased the secretion of sAPPα and this effect was blocked by protein kinase C (PKC) inhibitor calphostin C, but not by the classical estrogen receptor antagonist ICI 182,780. Meanwhile, 17β-estradiol did not alter the synthesis of cellular APP. Conclusion: The effect of 17β-estradiol on sAPPα secretion is likely mediated through the membrane binding sites, and needs molecular configuration specificity of the ligand. Furthermore, the action of the PKC dependent pathway might be involved in estrogen-induced sAPPα secretion.

The fluorescence-conjugated N-methyl-D-aspartate receptor-selective antagonist, BODIPY-conantokin-G, was employed to label N-methyl-D-aspartate receptors in living neurons derived from the visual cortex of embryonic rats. The fluorescent labeling was visualized and analysed using confocal microscopy and digital imaging techniques. BODIPY-conantokin-G binding sites were homogeneously distributed across somata four days after neurons (E17-20) were placed in culture. In five-day-old cultures, BODIPY-conantokin-G binding sites became clusters of fluorescently labeled spots which were arranged irregularly on somata and proximal neurites. Distal neurites displayed fluorescent labeling after 10-15 days in culture. Displacement experiments showed that spermine and unlabeled conantokin-G compete with BODIPY-conantokin-G labeling at the N-methyl-D-aspartate receptor-associated polyamine site. The N-methyl-D-aspartate receptor antagonist 2-amino-5-phosphonovaleric acid also depressed the labeling but with a weaker effect, probably due to interactions occurring between the N-methyl-D-aspartate receptor agonist binding site and the polyamine modulatory site. The fluorescent dyes FM 1-43 and FM 4-64 were used in double-labeling studies to compare the distribution of nerve terminals with that of BODIPY-conantokin-G binding sites. BODIPY-conantokin-G binding clusters were associated with presynaptic nerve terminals while isolated BODIPY-conantokin-G binding sites were not always opposed to terminals. The aggregation of receptors to form clusters may lead to the functional formation of excitatory synapses. To investigate whether modulation of membrane potentials affected the formation of N-methyl-D-aspartate receptor clusters, cultured neurons were chronically treated for a week with either tetrodotoxin (to block membrane action potentials) or a high concentration of potassium to depolarize the membrane. While neurons in the tetrodotoxin-treated group showed a similar number of

Full Text Available Autophagic (type II cell death, characterized by the massive accumulation of autophagic vacuoles in the cytoplasm of cells, has been suggested to play pathogenetic roles in cerebral ischemia, brain trauma, and neurodegenerative disorders. 3,4-Methylenedioxymethamphetamine (MDMA or ecstasy is an illicit drug causing long-term neurotoxicity in the brain. Apoptotic (type I and necrotic (type III cell death have been implicated in MDMA-induced neurotoxicity, while the role of autophagy in MDMA-elicited neurotoxicity has not been investigated. The present study aimed to evaluate the occurrence and contribution of autophagy to neurotoxicity in cultured rat corticalneurons challenged with MDMA. Autophagy activation was monitored by expression of microtubule-associated protein 1 light chain 3 (LC3; an autophagic marker using immunofluorescence and western blot analysis. Here, we demonstrate that MDMA exposure induced monodansylcadaverine (MDC- and LC3B-densely stained autophagosome formation and increased conversion of LC3B-I to LC3B-II, coinciding with the neurodegenerative phase of MDMA challenge. Autophagy inhibitor 3-methyladenine (3-MA pretreatment significantly attenuated MDMA-induced autophagosome accumulation, LC3B-II expression, and ameliorated MDMA-triggered neurite damage and neuronal death. In contrast, enhanced autophagy flux by rapamycin or impaired autophagosome clearance by bafilomycin A1 led to more autophagosome accumulation in neurons and aggravated neurite degeneration, indicating that excessive autophagosome accumulation contributes to MDMA-induced neurotoxicity. Furthermore, MDMA induced phosphorylation of AMP-activated protein kinase (AMPK and its downstream unc-51-like kinase 1 (ULK1, suggesting the AMPK/ULK1 signaling pathway might be involved in MDMA-induced autophagy activation.

Although the MAP kinase-interacting kinases (MNKs) have been known for >15 years, their roles in the regulation of protein synthesis have remained obscure. Here, we explore the involvement of the MNKs in brain-derived neurotrophic factor (BDNF)-stimulated protein synthesis in corticalneurons from mice. Using a combination of pharmacological and genetic approaches, we show that BDNF-induced upregulation of protein synthesis requires MEK/ERK signaling and the downstream kinase, MNK1, which phosphorylates eukaryotic initiation factor (eIF) 4E. Translation initiation is mediated by the interaction of eIF4E with the m(7)GTP cap of mRNA and with eIF4G. The latter interaction is inhibited by the interactions of eIF4E with partner proteins, such as CYFIP1, which acts as a translational repressor. We find that BDNF induces the release of CYFIP1 from eIF4E, and that this depends on MNK1. Finally, using a novel combination of BONCAT and SILAC, we identify a subset of proteins whose synthesis is upregulated by BDNF signaling via MNK1 in neurons. Interestingly, this subset of MNK1-sensitive proteins is enriched for functions involved in neurotransmission and synaptic plasticity. Additionally, we find significant overlap between our subset of proteins whose synthesis is regulated by MNK1 and those encoded by known FMRP-binding mRNAs. Together, our data implicate MNK1 as a key component of BDNF-mediated translational regulation in neurons.

Chronic alcohol consumption may cause neurodevelopmental and neurodegenerative disorders. Alcohol neurotoxicity is associated with the production of acetaldehyde and reactive oxygen species that induce oxidative DNA damage. However, the molecular mechanisms by which ethanol disturbs the DNA damage response (DDR), resulting in a defective DNA repair, remain unknown. Here, we have used cultured primary corticalneurons exposed to 50 or 100 mM ethanol for 7 days to analyze the ethanol-induced DDR. Ethanol exposure produced a dose-dependent generation of double strand breaks and the formation of DNA damage foci immunoreactive for the histone γH2AX, a DNA damage marker, and for the ubiquitylated H2A, which is involved in chromatin remodeling at DNA damage sites. Importantly, these DNA damage foci failed to recruit the protein 53BP1, a crucial DNA repair factor. This effect was associated with a drop in 53BP1 mRNA and protein levels and with an inhibition of global transcription. Moreover, ethanol-exposed neurons treated with ionizing radiation (2 Gy) also failed to recruit 53BP1 at DNA damage foci and exhibited a greater vulnerability to DNA lesions than irradiated control neurons. Our results support that defective DNA repair, mediated by the deficient expression and recruitment of 53BP1 to DNA damage sites, represents a novel mechanism involved in ethanol neurotoxicity. The design of therapeutic strategies that increase or stabilize 53BP1 levels might potentially promote DNA repair and partially compensate alcohol neurotoxicity.

Brain ischemic preconditioning (PC) provides vital insights into the endogenous protection against stroke. Genomic and epigenetic responses to PC condition the brain into a state of ischemic tolerance. Notably, PC induces the elevation of histone acetylation, consistent with evidence that histone deacetylase (HDAC) inhibitors protect the brain from ischemic injury. However, less is known about the specific roles of HDACs in this process. HDAC3 has been implicated in several neurodegenerative conditions. Deletion of HDAC3 confers protection against neurotoxicity and neuronal injury. Here, we hypothesized that inhibition of HDAC3 may contribute to the neuronal survival elicited by PC. To address this notion, PC and transient middle cerebral artery occlusion (MCAO) were conducted in Sprague-Dawley rats. Additionally, primary cultured corticalneurons were used to identify the modulators and effectors of HDAC3 involved in PC. We found that nuclear localization of HDAC3 was significantly reduced following PC in vivo and in vitro. Treatment with the HDAC3-specific inhibitor, RGFP966, mimicked the neuroprotective effects of PC 24 h and 7 days after MCAO, causing a reduced infarct volume and less Fluoro-Jade C staining. Improved functional outcomes were observed in the neurological score and rotarod test. We further showed that attenuated recruitment of HDAC3 to promoter regions following PC potentiates transcriptional initiation of genes including Hspa1a, Bcl2l1, and Prdx2, which may underlie the mechanism of protection. In addition, PC-activated calpains were implicated in the cleavage of HDAC3. Pretreatment with calpeptin blockaded the attenuated nuclear distribution of HDAC3 and the protective effect of PC in vivo. Collectively, these results demonstrate that the inhibition of HDAC3 preconditions the brain against ischemic insults, indicating a new approach to evoke endogenous protection against stroke. PMID:27965534

Full Text Available Brain ischemic preconditioning (PC provides vital insights into the endogenous protection against stroke. Genomic and epigenetic responses to PC condition the brain into a state of ischemic tolerance. Notably, PC induces the elevation of histone acetylation, consistent with evidence that histone deacetylase (HDAC inhibitors protect the brain from ischemic injury. However, less is known about the specific roles of HDACs in this process. HDAC3 has been implicated in several neurodegenerative conditions. Deletion of HDAC3 confers protection against neurotoxicity and neuronal injury. Here, we hypothesized that inhibition of HDAC3 may contribute to the neuronal survival elicited by PC. To address this notion, PC and transient middle cerebral artery occlusion (MCAO were conducted in Sprague-Dawley rats. Additionally, primary cultured corticalneurons were used to identify the modulators and effectors of HDAC3 involved in PC. We found that nuclear localization of HDAC3 was significantly reduced following PC in vivo and in vitro. Treatment with the HDAC3-specific inhibitor, RGFP966, mimicked the neuroprotective effects of PC 24 h and 7 d after MCAO, causing a reduced infarct volume and less Fluoro-Jade C staining. Improved functional outcomes were observed in the neurological score and rotarod test. We further showed that attenuated recruitment of HDAC3 to promoter regions following PC potentiates transcriptional initiation of genes including Hspa1a, Bcl2l1, and Prdx2, which may underlie the mechanism of protection. In addition, PC-activated calpains were implicated in the cleavage of HDAC3. Pretreatment with calpeptin blockaded the attenuated nuclear distribution of HDAC3 and the protective effect of PC in vivo. Collectively, these results demonstrate that the inhibition of HDAC3 preconditions the brain against ischemic insults, indicating a new approach to evoke endogenous protection against stroke.

Full Text Available This research was done for the Summer Internship in Neural Engineering (SINE during a three month period, June 2008 until the end of August 2008. The SINE program is affiliated with the Sensory Motor Performance Program (SMPP at the Rehabilitation Institute of Chicago (RIC and the Biomedical Engineering program at Northwestern University. I worked in the Neuralplasticity laboratory, which is a part of the SMPP located at the RIC. I worked under Dr. Stinear and Dr. Madhavan to test protocols developed by my advisors as candidate techniques for demonstrating ipsilateral connectivity between the lower limb motor cortex and spinal motor neurons. The goal of the research was to develop a candidate stimulation protocol to demonstrate ipsilateral connectivity in stroke patients between the lower limb motor cortex and spinal motor neurons.

The retrosplenial cortex (RSC) plays an important role in memory and spatial navigation. It shares functional similarities with the hippocampus, including the presence of place fields and lesion-induced impairments in spatial navigation, and the RSC is an important source of visual-spatial input to the hippocampus. Recently, the RSC has been the target of intense scrutiny among investigators of human memory and navigation. fMRI and lesion data suggest an RSC role in the ability to use landmarks to navigate to goal locations. However, no direct neurophysiological evidence of encoding navigational cues has been reported so the specific RSC contribution to spatial cognition has been uncertain. To examine this, we trained rats on a T-maze task in which the reward location was explicitly cued by a flashing light and we recorded RSC neurons as the rats learned. We found that RSC neurons rapidly encoded the light cue. Additionally, RSC neurons encoded the reward and its location, and they showed distinct firing patterns along the left and right trajectories to the goal. These responses may provide key information for goal-directed navigation, and the loss of these signals may underlie navigational impairments in subjects with RSC damage.

Full Text Available Williams syndrome (WS is a unique neurodevelopmental disorder with a specific behavioral and cognitive profile, which includes hyperaffiliative behavior, poor social judgment, and lack of social inhibition. Here we examined the morphology of basal dendrites on pyramidal neurons in the cortex of two rare adult subjects with WS. Specifically, we examined two areas in the prefrontal cortex (PFC—the frontal pole (Brodmann area 10 and the orbitofrontal cortex (Brodmann area 11—and three areas in the motor, sensory, and visual cortex (BA 4, BA 3-1-2, BA 18. The findings suggest that the morphology of basal dendrites on the pyramidal neurons is altered in the cortex of WS, with differences that were layer-specific, more prominent in PFC areas, and displayed an overall pattern of dendritic organization that differentiates WS from other disorders. In particular, and unlike what was expected based on typically developing brains, basal dendrites in the two PFC areas did not display longer and more branched dendrites compared to motor, sensory and visual areas. Moreover, dendritic branching, dendritic length, and the number of dendritic spines differed little within PFC and between the central executive region (BA 10 and BA 11 that is part of the orbitofrontal region involved into emotional processing. In contrast, the relationship between the degree of neuronal branching in supra- versus infra-granular layers was spared in WS. Although this study utilized tissue held in formalin for a prolonged period of time and the number of neurons available for analysis was limited, our findings indicate that WS cortex, similar to that in other neurodevelopmental disorders such as Down syndrome, Rett syndrome, Fragile X, and idiopathic autism, has altered morphology of basal dendrites on pyramidal neurons, which appears more prominent in selected areas of the PFC. Results were examined from developmental perspectives and discussed in the context of other

Williams syndrome (WS) is a unique neurodevelopmental disorder with a specific behavioral and cognitive profile, which includes hyperaffiliative behavior, poor social judgment, and lack of social inhibition. Here we examined the morphology of basal dendrites on pyramidal neurons in the cortex of two rare adult subjects with WS. Specifically, we examined two areas in the prefrontal cortex (PFC)-the frontal pole (Brodmann area 10) and the orbitofrontal cortex (Brodmann area 11)-and three areas in the motor, sensory, and visual cortex (BA 4, BA 3-1-2, BA 18). The findings suggest that the morphology of basal dendrites on the pyramidal neurons is altered in the cortex of WS, with differences that were layer-specific, more prominent in PFC areas, and displayed an overall pattern of dendritic organization that differentiates WS from other disorders. In particular, and unlike what was expected based on typically developing brains, basal dendrites in the two PFC areas did not display longer and more branched dendrites compared to motor, sensory and visual areas. Moreover, dendritic branching, dendritic length, and the number of dendritic spines differed little within PFC and between the central executive region (BA 10) and BA 11 that is part of the orbitofrontal region involved into emotional processing. In contrast, the relationship between the degree of neuronal branching in supra- versus infra-granular layers was spared in WS. Although this study utilized tissue held in formalin for a prolonged period of time and the number of neurons available for analysis was limited, our findings indicate that WS cortex, similar to that in other neurodevelopmental disorders such as Down syndrome, Rett syndrome, Fragile X, and idiopathic autism, has altered morphology of basal dendrites on pyramidal neurons, which appears more prominent in selected areas of the PFC. Results were examined from developmental perspectives and discussed in the context of other neurodevelopmental disorders

Glutamate and nitric oxide (NO) are important regulators of dendrite and axon development in the central nervous system. Excess glutamatergic stimulation is a feature of many pathological conditions and manifests in neuronal atrophy and shrinkage with eventual neurodegeneration and cell death. Here we demonstrate that treatment of cultured primary cortical rat neurons for 24h with glutamate (500μM) or N-methyl-d-aspartate (NMDA) (100-500μM) combined with glycine suppresses neurite outgrowth. A similar reduction of neurite outgrowth was observed with the NO precursor l-arginine and NO donor sodium nitroprusside (SNP) (100 and 300μM). The NMDA-receptor (NMDA-R) antagonists ketamine and MK-801 (10nM) counteracted the NMDA/glycine-induced reduction in neurite outgrowth and the neuronal NO synthase (nNOS) inhibitor 1-[2-(trifluoromethyl)phenyl] imidazole (TRIM) (100nM) counteracted both the NMDA/glycine and l-arginine-induced decreases in neurite outgrowth. Furthermore, targeting soluble guanylate cyclase (sGC), a downstream target of NO, with the sGC inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) (10μM) also protected against l-arginine-induced decreases in neurite outgrowth. Since the NMDA-R is functionally coupled to nNOS via the postsynaptic protein 95kDa (PSD-95), inhibitors of the PSD-95/nNOS interaction were tested for their ability to protect against glutamate-induced suppression in neurite outgrowth. Treatment with the small-molecule inhibitors of the PSD-95/nNOS interface 2-((1H-benzo[d] [1,2,3]triazol-5-ylamino) methyl)-4,6-dichlorophenol (IC87201) (10 and 100nM) and 4-(3,5-dichloro-2-hydroxy-benzylamino)-2-hydroxybenzoic acid (ZL-006) (10 and 100nM) attenuated NMDA/glycine-induced decreases in neurite outgrowth. These data support the hypothesis that targeting the NMDA-R/PSD-95/nNOS interaction downstream of NMDA-R promotes neurotrophic effects by preventing neurite shrinkage in response to excess glutamatergic stimulation. The PSD-95/n

We used hierarchical tree clustering to derive a functional organizational chart of 52 human cortical areas (26 per hemisphere) from zero-lag correlations calculated between single-voxel, prewhitened, resting-state BOLD fMRI time series in 18 subjects. No special "resting-state networks" were identified. There were four major features in the resulting tree (dendrogram). First, there was a strong clustering of homotopic, left-right hemispheric areas. Second, cortical areas were concatenated in multiple, partially overlapping clusters. Third, the arrangement of the areas revealed a layout that closely resembled the actual layout of the cerebral cortex, namely an orderly progression from anterior to posterior. And fourth, the layout of the cortical areas in the tree conformed to principles of efficient, compact layout of components proposed by Cherniak. Since the tree was derived on the basis of the strength of neural correlations, these results document an orderly relation between functional interactions and layout, i.e., between structure and function.

BACKGROUND: Cardiocerebrovascular diseases induced cerebral circulation insufficiency and senile vascular dementia can result in ischemic/hypoxic apoptosis of central neurons, which we should pay more attention to and prevent and treat as early as possible. Traditional Chinese medicine possesses the unique advantage in this field. Polygonatum, a Chinese herb for invigorating qi, may play a role against the hypoxic apoptosis of brain neurons.OBJECTIVE: To observe the protective effect of polygonatum polysaccharide on hypoxia-induced apoptosis and necrosis in cerebral corticalneurons cultured in vitro.DESIGN: A comparative experiment.SETTING: Laboratory of Cell Biology, Institute of Basic Medical Sciences, Jiangxi Provincial Academy of Traditional Chinese Medicine.MATERIALS: The experiment was carried out in the Laboratory of Cell Biology, Institute of Basic Medical Sciences, Jiangxi Provincial Academy of Traditional Chinese Medicine from November 2003 to April 2005.Totally 218 Wistar rats (male or female) of clean degree within 24 hours after birth were purchased from the animal center of Jiangxi Medical College (certification number was 021-97-03).METHODS: ① Preparation of cerebral corticalneurons of rats: The cerebral cortical tissues were isolated from the Wistar rats within 24 hours after birth, and prepared to single cell suspension, and the cerebral corticalneurons of neonatal rats were in vitro cultured in serum free medium with Neurobasal plus B27Supplement. ② Observation on the non-toxic dosage of polygonatum polysaccharide on neurons: After the neurons were cultured for 4 days, polygonatum polysaccharide of different dosages (1-20 g/L) was added for continuous culture for 48 hours, the toxicity and non-toxic dosage of polygonatum polysaccharide on neurons were observed and detected with trypan blue staining. ③ Grouping: After hypoxia/reoxygenation,the cultured neurons were divided into normal control group, positive apoptotic group and polygonatum

Firstly, the possible MKs protection against mitochondrial dysfunction caused by oxidative stress was tested. Mitochondrial function was analyzed by MTT, also correlated with neurons survival measurements (Varming et al., 1996. MKs, at the two chosen concentrations, were co-incubated with H2O2 (200 µM for 12h, and viability assays were performed. Results demonstrated that the viability of neurons treated with the oxidant decreased a 31.6 ± 2.0% (p 2O2 insults. TRMR test reveals a diminution of 33.6 ± 4.3% (p 2O2 treatments in neurons elevated ROS production in a 20.0 ± 2.5% (p 2O2 as previously described and ROS levels were measured. A reduction of ROS levels regarding the oxidant treatment was observed in MKs H, J, F and G treatments. In physiological conditions, low concentrations of H2O2 are transformed to water and molecular oxygen by GSH–peroxidase, with GSH as a proton donor. But when H2O2 amounts are high, they are instead eliminated by CAT. GSH is one of the antioxidant mitochondrial systems of protection against oxidative damage (Bains and Shaw, 1997. So to conclude the antioxidant research, MKs effects over GSH and CAT were evaluated. GSH is the main intracellular thiol in cells (Zampagni et al., 2012 and a thiol tracker was used to evaluate it. 12h H2O2 incubation produces a GSH level reduction of 25.8 ± 3.1% (p 2O2, as detailed above, and only MK J increased its levels to a 92.5 ± 9.4% (p = 0.048, achieving GSH basal amounts. Moreover the oxidation treatment decreases CAT activity in neurons in a 24.4 ± 5.5% (p < 0.01 however, the co-incubation with MKs increased CAT activity. MKs J, L and G treatments produced a significant elevation with a complete reestablishment of the activity. Neurons consume an elevated percentage of total body oxygen and consequently they are one of the most vulnerable cell populations to oxidative stress, which plays an important role in neurodegenerative pathology . After MKs evaluation in neurons under oxidative

Full Text Available Recurrent/moderate (R/M hypoglycemia is common in type 1 diabetes patients. Moderate hypoglycemia is not life-threatening, but if experienced recurrently it may present several clinical complications. Activated PARP-1 consumes cytosolic NAD, and because NAD is required for glycolysis, hypoglycemia-induced PARP-1 activation may render cells unable to use glucose even when glucose availability is restored. Pyruvate, however, can be metabolized in the absence of cytosolic NAD. We therefore hypothesized that pyruvate may be able to improve the outcome in diabetic rats subjected to insulin-induced R/M hypoglycemia by terminating hypoglycemia with glucose plus pyruvate, as compared with delivering just glucose alone. In an effort to mimic juvenile type 1 diabetes the experiments were conducted in one-month-old young rats that were rendered diabetic by streptozotocin (STZ, 50mg/kg, i.p. injection. One week after STZ injection, rats were subjected to moderate hypoglycemia by insulin injection (10 U/kg, i.p. without anesthesia for five consecutive days. Pyruvate (500 mg/kg was given by intraperitoneal injection after each R/M hypoglycemia. Three hours after last R/M hypoglycemia, zinc accumulation was evaluated. Three days after R/M hypoglycemia, neuronal death, oxidative stress, microglial activation and GSH concentrations in the cerebral cortex were analyzed. Sparse neuronal death was observed in the cortex. Zinc accumulation, oxidative injury, microglial activation and GSH loss in the cortex after R/M hypoglycemia were all reduced by pyruvate injection. These findings suggest that when delivered alongside glucose, pyruvate may significantly improve the outcome after R/M hypoglycemia by circumventing a sustained impairment in neuronal glucose utilization resulting from PARP-1 activation.

Full Text Available DNA methylation is capable of modulating coordinate expression of large numbers of genes across many different pathways, and may therefore warrant investigation for their potential role between genes and disease phenotype. In a rare set of monozygotic twins discordant for Alzheimer's disease (AD, significantly reduced levels of DNA methylation were observed in temporal neocortex neuronal nuclei of the AD twin. These findings are consistent with the hypothesis that epigenetic mechanisms may mediate at the molecular level the effects of life events on AD risk, and provide, for the first time, a potential explanation for AD discordance despite genetic similarities.

Correlating in vivo imaging of neurons and their synaptic connections with electron microscopy combines dynamic and ultrastructural information. Here we describe a semi-automated technique whereby volumes of brain tissue containing axons and dendrites, previously studied in vivo, are subsequently imaged in three dimensions with focused ion beam scanning electron microcopy. These neurites are then identified and reconstructed automatically from the image series using the latest segmentation algorithms. The fast and reliable imaging and reconstruction technique avoids any specific labeling to identify the features of interest in the electron microscope, and optimises their preservation and staining for 3D analysis. PMID:23468982

Full Text Available Correlating in vivo imaging of neurons and their synaptic connections with electron microscopy combines dynamic and ultrastructural information. Here we describe a semi-automated technique whereby volumes of brain tissue containing axons and dendrites, previously studied in vivo, are subsequently imaged in three dimensions with focused ion beam scanning electron microcopy. These neurites are then identified and reconstructed automatically from the image series using the latest segmentation algorithms. The fast and reliable imaging and reconstruction technique avoids any specific labeling to identify the features of interest in the electron microscope, and optimises their preservation and staining for 3D analysis.

Full Text Available The human prefrontal cortex has been considered different in several aspects and relatively enlarged compared to the rest of the cortical areas. Here we determine whether the white and gray matter of the prefrontal portion of the human cerebral cortex have similar or different cellular compositions relative to the rest of the cortical regions by applying the Isotropic Fractionator to analyze the distribution of neurons along the entire anteroposterior axis of the cortex, and its relationship with the degree of gyrification, number of neurons under the cortical surface, and other parameters. The prefrontal region shares with the remainder of the cerebral cortex (except for occipital cortex the same relationship between cortical volume and number of neurons. In contrast, both occipital and prefrontal areas vary from other cortical areas in their connectivity through the white matter, with a systematic reduction of cortical connectivity through the white matter and an increase of the mean axon caliber along the anteroposterior axis. These two parameters explain local differences in the distribution of neurons underneath the cortical surface. We also show that local variations in cortical folding are neither a function of local numbers of neurons nor of cortical thickness, but correlate with properties of the white matter, and are best explained by the folding of the white matter surface. Our results suggest that the human cerebral cortex is divided in two zones (occipital and non-occipital that differ in how neurons distributed across their grey matter volume and in three zones (prefrontal, occipital, and non-occipital that differ in how neurons are connected through the white matter. Thus, the human prefrontal cortex has the largest fraction of neuronal connectivity through the white matter and the smallest average axonal caliber in the white matter within the cortex, although its neuronal composition fits the pattern found for other, non

Huntington disease (HD), a neurodegenerative disorder caused by CAG repeat expansion in the gene encoding huntingtin, predominantly affects the striatum, especially the spiny projection neurons (SPN). The striatum receives excitatory input from cortex and thalamus, and the role of the former has been well-studied in HD. Here, we report that mutated huntingtin alters function of thalamostriatal connections. We used a novel thalamostriatal (T-S) coculture and an established corticostriatal (C-S) coculture, generated from YAC128 HD and WT (FVB/NJ background strain) mice, to investigate excitatory neurotransmission onto striatal SPN. SPN in T-S coculture from WT mice showed similar mini-excitatory postsynaptic current (mEPSC) frequency and amplitude as in C-S coculture; however, both the frequency and amplitude were significantly reduced in YAC128 T-S coculture. Further investigation in T-S coculture showed similar excitatory synapse density in WT and YAC128 SPN dendrites by immunostaining, suggesting changes in total dendritic length or probability of release as possible explanations for mEPSC frequency changes. Synaptic N-methyl-D-aspartate receptor (NMDAR) current was similar, but extrasynaptic current, associated with cell death signaling, was enhanced in YAC128 SPN in T-S coculture. Employing optical stimulation of cortical versus thalamic afferents and recording from striatal SPN in brain slice, we found increased glutamate release probability and reduced AMPAR/NMDAR current ratios in thalamostriatal synapses, most prominently in YAC128. Enhanced extrasynaptic NMDAR current in YAC128 SPN was apparent with both cortical and thalamic stimulation. We conclude that thalamic afferents to the striatum are affected early, prior to an overt HD phenotype; however, changes in NMDAR localization in SPN are independent of the source of glutamatergic input.

Full Text Available Angong Niuhuang pill, a Chinese materia medica preparation, can improve neurological functions after acute ischemic stroke. Because of its inconvenient application and toxic components (Cinnabaris and Realgar, we used transdermal enhancers to deliver Angong Niuhuang pill by modern technology, which expanded the safe dose range and clinical indications. In this study, Angong Niuhuang stickers administered at different point application doses (1.35, 2.7, and 5.4 g/kg were administered to the Dazhui (DU14, Qihai (RN6 and Mingmen (DU4 of rats with chronic cerebral ischemia, for 4 weeks. The Morris water maze was used to determine the learning and memory ability of rats. Hematoxylin-eosin staining and Nissl staining were used to observe neuronal damage of the cortex and hippocampal CA1 region in rats with chronic cerebral ischemia. The middle- and high-dose point application of Angong Niuhuang stickers attenuated neuronal damage in the cortex and hippocampal CA1 region, and improved the memory of rats with chronic cerebral ischemia with an efficacy similar to interventions by electroacupuncture at Dazhui (DU14, Qihai (RN6 and Mingmen (DU4. Our experimental findings indicate that point application with Angong Niuhuang stickers can improve cognitive function after chronic cerebral ischemia in rats and is neuroprotective with an equivalent efficacy to acupuncture.

Highlights: Black-Right-Pointing-Pointer 710 nm wavelength light (LED) has a protective effect in the stroke animal model. Black-Right-Pointing-Pointer We determined the effects of LED irradiation in vitro stroke model. Black-Right-Pointing-Pointer LED treatment promotes the neurite outgrowth through MAPK activation. Black-Right-Pointing-Pointer The level of synaptic markers significantly increased with LED treatment. Black-Right-Pointing-Pointer LED treatment protects cell death in the in vitro stroke model. -- Abstract: Objective: We previously reported that 710 nm Light-emitting Diode (LED) has a protective effect through cellular immunity activation in the stroke animal model. However, whether LED directly protects neurons suffering from neurodegeneration was entirely unknown. Therefore, we sought to determine the effects of 710 nm visible light irradiation on neuronal protection and neuronal outgrowth in an in vitro stroke model. Materials and methods: Primary cultured rat corticalneurons were exposed to oxygen-glucose deprivation (OGD) and reoxygenation and normal conditions. An LED array with a peak wavelength of 710 nm was placed beneath the covered culture dishes with the room light turned off and were irradiated accordingly. LED treatments (4 min at 4 J/cm{sup 2} and 50 mW/cm{sup 2}) were given once to four times within 8 h at 2 h intervals for 7 days. Mean neurite density, mean neurite diameter, and total fiber length were also measured after microtubule associated protein 2 (MAP2) immunostaining using the Axio Vision program. Synaptic marker expression and MAPK activation were confirmed by Western blotting. Results: Images captured after MAP2 immunocytochemistry showed significant (p < 0.05) enhancement of post-ischemic neurite outgrowth with LED treatment once and twice a day. MAPK activation was enhanced by LED treatment in both OGD-exposed and normal cells. The levels of synaptic markers such as PSD 95, GAP 43, and synaptophysin significantly

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder that results in motor, cognitive and psychiatric abnormalities. Dysfunction in neuronal processing between the cortex and the basal ganglia is fundamental to the onset and progression of the HD phenotype. The corticosubthalamic hyperdirect pathway plays a crucial role in motor selection and blockade of neuronal activity in the subthalamic nucleus (STN) results in hyperkinetic movement abnormalities, similar to the motor symptoms associated with HD. The aim of the present study was to examine whether changes in the fidelity of information transmission between the cortex and the STN emerge as a function of phenotypic severity in the YAC128 mouse model of HD. We obtained in vivo extracellular recordings in the STN and concomitant electrocorticogram (ECoG) recordings during discrete brain states that reflected global cortical network synchronization or desynchronization. At early ages in YAC128 mice, both the cortex and the STN exhibited patterns of hyperexcitability. As symptom severity progressed, cortical entrainment of STN activity was disrupted and there was an increase in the proportion of non-oscillating, tonically firing STN neurons that were less phase-locked to cortical activity. Concomitant to the dissipation of STN entrainment, there was a reduction in the evoked response of STN neurons to focal cortical stimulation. The spontaneous discharge of STN neurons in YAC128 mice also decreased with age and symptom severity. These results indicate dysfunction in the flow of information within the corticosubthalamic circuit and demonstrate progressive age-related disconnection of the hyperdirect pathway in a transgenic mouse model of HD.

Ecstasy, 3,4-methylenedioxymetamphetamine (MDMA), is a recreational drug used among adolescents, including young pregnant women. MDMA passes the placental barrier and may therefore influence fetal development. The aim was to investigate the direct effect of MDMA on cortical cells using dissociated...... CNS cortex of rat embryos, E17. The primary culture was exposed to a single dose of MDMA and collected 5 days later. MDMA caused a dramatic, dose-dependent (100 and 400 muM) decrease in nestin-positive stem cell density, as well as a significant reduction (400 muM) in NeuN-positive cells. By q......PCR, MDMA (200 muM) caused a significant decrease in mRNA expression of the 5HT3 receptor, dopamine D(1) receptor, and glutamate transporter EAAT2-1, as well as an increase in mRNA levels of the NMDA NR1 receptor subunit and the 5HT(1A) receptor. In conclusion, MDMA caused a marked reduction in stem cells...

Nefiracetam is a new pyrrolidone nootropic drug being developed for the treatment of Alzheimer's type and post-stroke vascular-type dementia. In the brain of Alzheimer's disease patients, down-regulation of both cholinergic and glutamatergic systems has been found and is thought to play an important role in impairment of cognition, learning and memory. We have previously shown that the activity of neuronal nicotinic acetylcholine receptors is potently augmented by nefiracetam. The present study was undertaken to elucidate the mechanism of action of nefiracetam on glutamatergic receptors. Currents were recorded from rat corticalneurons in long-term primary culture using the whole-cell patch-clamp technique at a holding potential of -70 mV in Mg2+-free solutions. N-Methyl-D-aspartate (NMDA)-evoked currents were greatly and reversibly potentiated by bath application of nefiracetam resulting in a bell-shaped dose-response curve. The minimum effective nefiracetam concentration was 1 nM, and the maximum potentiation to 170% of the control was produced at 10 nM. Nefiracetam potentiation occurred at high NMDA concentrations that evoked the saturated response, and in a manner independent of NMDA concentrations ranging from 3 to 1,000 microM. Glycine at 3 microM potentiated NMDA currents but this effect was attenuated with an increasing concentration of nefiracetam from 1 to 10,000 nM. 7-Chlorokynurenic acid at 1 microM prevented nefiracetam from potentiating NMDA currents. Nefiracetam at 10 nM shifted the dose-response relationship for the 7-chlorokynurenic acid inhibition of NMDA currents in the direction of higher concentrations. Alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid- and kainate-induced currents were not significantly affected by application of 10 nM nefiracetam. It was concluded that nefiracetam potentiated NMDA currents through interactions with the glycine binding site of the NMDA receptor.

Full Text Available Excessive "excitotoxic" accumulation of Ca(2+ and Zn(2+ within neurons contributes to neurodegeneration in pathological conditions including ischemia. Putative early targets of these ions, both of which are linked to increased reactive oxygen species (ROS generation, are mitochondria and the cytosolic enzyme, NADPH oxidase (NOX. The present study uses primary corticalneuronal cultures to examine respective contributions of mitochondria and NOX to ROS generation in response to Ca(2+ or Zn(2+ loading. Induction of rapid cytosolic accumulation of either Ca(2+ (via NMDA exposure or Zn(2+ (via Zn(2+/Pyrithione exposure in 0 Ca(2+ caused sharp cytosolic rises in these ions, as well as a strong and rapid increase in ROS generation. Inhibition of NOX activation significantly reduced the Ca(2+-induced ROS production with little effect on the Zn(2+- triggered ROS generation. Conversely, dissipation of the mitochondrial electrochemical gradient increased the cytosolic Ca(2+ or Zn(2+ rises caused by these exposures, consistent with inhibition of mitochondrial uptake of these ions. However, such disruption of mitochondrial function markedly suppressed the Zn(2+-triggered ROS, while partially attenuating the Ca(2+-triggered ROS. Furthermore, block of the mitochondrial Ca(2+ uniporter (MCU, through which Zn(2+ as well as Ca(2+ can enter the mitochondrial matrix, substantially diminished Zn(2+ triggered ROS production, suggesting that the ROS generation occurs specifically in response to Zn(2+ entry into mitochondria. Finally, in the presence of the sulfhydryl-oxidizing agent 2,2'-dithiodipyridine, which impairs Zn(2+ binding to cytosolic metalloproteins, far lower Zn(2+ exposures were able to induce mitochondrial Zn(2+ uptake and consequent ROS generation. Thus, whereas rapid acute accumulation of Zn(2+ and Ca(2+ each can trigger injurious ROS generation, Zn(2+ entry into mitochondria via the MCU may do so with particular potency. This may be of particular

Background Senile plaques and neurofibrillary tangles (NFTs) represent two of the major histopathological hallmarks of Alzheimer's disease (AD). The plaques are primarily composed of aggregated amyloid β (Aβ) peptides. The processing of amyloid-β precursor protein (AβPP) in okadaic acid (OA)-induced tau phosphorylation primary neurons was studied.Methods Primary cultures of rat brain corticalneurons were treated with OA and β-secretase inhibitor. Neurons' viability was measured. AβPP processing was examined by immunocytochemistry and Western blotting with specific antibodies against the AβPP-N-terminus (NT) and AβPP-C-terminus (CT).Results Ten nrnol/L OA had a time-dependent suppression effect on primary neurons' viability. The suppression effect was alleviated markedly by pretreatment with β-secretase inhibitor. After OA treatment, both AβPP and β-C-terminal fragment (βCTF) were significantly increased in neurons. AβPP level was increased further in neurons pretreated with β-secretase inhibitor.Conclusions In OA-induced tau phosphorylation cell model, inhibition of β-secretase may protect neurons from death induced by OA. Because of increased accumulation of AβPP in neurons after OA treatment, more AβPP turns to be cleaved by β-secretase, producing neurotoxic βCTF. As a potential effective therapeutic target, β-secretase is worth investigating further.

CSP-1103 (formerly CHF5074) has been shown to reverse memory impairment and reduce amyloid plaque as well as inflammatory microglia activation in preclinical models of Alzheimer’s disease. Moreover, it was found to improve cognition and reduce brain inflammation in patients with mild cognitive impairment. Recent evidence suggests that CSP-1103 acts through a single molecular target, the amyloid precursor protein intracellular domain (AICD), a transcriptional regulator implicated in inflammation and apoptosis. We here tested the possible anti-apoptotic and neuroprotective activity of CSP-1103 in a cell-based model of post-ischemic injury, wherein the primary mouse corticalneurons were exposed to oxygen-glucose deprivation (OGD). When added after OGD, CSP-1103 prevented the apoptosis cascade by reducing cytochrome c release and caspase-3 activation and the secondary necrosis. Additionally, CSP-1103 limited earlier activation of p38 and nuclear factor κB (NF-κB) pathways. These results demonstrate that CSP-1103 is neuroprotective in a model of post-ischemic brain injury and provide further mechanistic insights as regards its ability to reduce apoptosis and potential production of pro-inflammatory cytokines. In conclusion, these findings suggest a potential use of CSP-1103 for the treatment of brain ischemia. PMID:28106772

Full Text Available The phosphorylation of Amyloid Precursor Protein (APP at Thr668 plays a key role in APP metabolism that is highly relevant to AD. The c-Jun-N-terminal kinase (JNK, glycogen synthase kinase-3β (GSK-3β and cyclin-dependent kinase 5 (Cdk5 can all be responsible for this phosphorylation. These kinases are activated by excitotoxic stimuli fundamental hallmarks of AD. The exposure of corticalneurons to a high dose of NMDA (100 μM for 30’-45’ led to an increase of P-APP Thr668. During NMDA stimulation APP hyperphosphorylation has to be assigned to GSK-3β activity, since addition of L803-mts, a substrate competitive inhibitor of GSK-3β reduced APP phosphorylation induced by NMDA. On the contrary, inhibition of JNK and Cdk5 with D-JNKI1 and Roscovitine respectively did not prevent NMDA-induced P-APP increase. These data show a tight connection, in excitotoxic conditions, between APP metabolism and the GSK-3β signaling pathway.

Full Text Available Background: Experimental evidence supports the neuroprotective properties of lithium, with implications for the treatment and prevention of dementia and other neurodegenerative disorders. Lithium modulates critical intracellular pathways related to neurotrophic support, inflammatory response, autophagy and apoptosis. There is additional evidence indicating that lithium may also affect membrane homeostasis. Objective: To investigate the effect of lithium on cytosolic phospholipase A2 (PLA2 activity, a key player on membrane phospholipid turnover which has been found to be reduced in blood and brain tissue of patients with Alzheimer’s disease (AD. Methods: Primary cultures of cortical and hippocampal neurons were treated for 7 days with different concentrations of lithium chloride (0.02 mM, 0.2 mM and 2 mM. A radio-enzymatic assay was used to determine the total activity of PLA2 and two PLA2 subtypes: cytosolic calcium-dependent (cPLA2; and calcium-independent (iPLA2. Results: cPLA2 activity increased by 82% (0.02 mM; p = 0.05 and 26% (0.2 mM; p = 0.04 in corticalneurons and by 61% (0.2 mM; p = 0.03 and 57% (2 mM; p = 0.04 in hippocampal neurons. iPLA2 activity was increased by 7% (0.2 mM; p = 0.04 and 13% (2 mM; p = 0.05 in corticalneurons and by 141% (0.02 mM; p = 0.0198 in hippocampal neurons. Conclusion: long-term lithium treatment increases membrane phospholipid metabolism in neurons through the activation of total, c- and iPLA2. This effect is more prominent at sub-therapeutic concentrations of lithium, and the activation of distinct cytosolic PLA2 subtypes is tissue specific, i.e., iPLA2 in hippocampal neurons, and cPLA2 in corticalneurons. Because PLA2 activities are reported to be reduced in Alzheimer’s disease (AD and bipolar disorder (BD, the present findings provide a possible mechanism by which long-term lithium treatment may be useful in the prevention of the disease.

Alzheimer's disease is a progressive neurodegenerative disease that has links with other conditions that can often be modified by dietary and life-style interventions. In particular, coconut oil has received attention as having potentially having benefits in lessening the cognitive deficits associated with Alzheimer's disease. In a recent report, we showed that neuron survival in cultures co-treated with coconut oil and Aβ was rescued compared to cultures exposed only to Aβ. Here we investigated treatment with Aβ for 1, 6 or 24 h followed by addition of coconut oil for a further 24 h, or treatment with coconut oil for 24 h followed by Aβ exposure for various periods. Neuronal survival and several cellular parameters (cleaved caspase 3, synaptophysin labeling and ROS) were assessed. In addition, the influence of these treatments on relevant signaling pathways was investigated with Western blotting. In terms of the treatment timing, our data indicated that coconut oil rescues cells pre-exposed to Aβ for 1 or 6 h, but is less effective when the pre-exposure has been 24 h. However, pretreatment with coconut oil prior to Aβ exposure showed the best outcomes. Treatment with octanoic or lauric acid also provided protection against Aβ, but was not as effective as the complete oil. The coconut oil treatment reduced the number of cells with cleaved caspase and ROS labeling, as well as rescuing the loss of synaptophysin labeling observed with Aβ treatment. Treatment with coconut oil, as well as octanoic, decanoic and lauric acids, resulted in a modest increase in ketone bodies compared to controls. The biochemical data suggest that Akt and ERK activation may contribute to the survival promoting influence of coconut oil. This was supported by observations that a PI3-Kinase inhibitor blocked the rescue effect of CoOil on Aβ amyloid toxicity. Further studies into the mechanisms of action of coconut oil and its constituent medium chain fatty acids are warranted.

Full Text Available The marine habitat provides a large number of structurally-diverse bioactive compounds for drug development. Marine sponges have been studied over many years and are found to be a rich source of these bioactive chemicals. This study is focused on the evaluation of the activity of six diterpene derivatives isolated from Spongionella sp. on mitochondrial function using an oxidative in vitro stress model. The test compounds include the Gracilins (A, H, K, J and L and tetrahydroaplysulphurin-1. Compounds were co-incubated with hydrogen peroxide for 12 hours to determine their protective capacities and their effect on markers of apoptosis and Nrf2/ARE pathways was evaluated. Results conclude that Gracilins preserve neurons against oxidative damage, and that in particular, tetrahydroaplysulphurin-1 shows a complete neuroprotective activity. Oxidative stress is linked to mitochondrial dysfunction and consequently to neurodegenerative disorders like Parkinson and Alzheimer diseases, Friedreich ataxia or Amyotrophic lateral sclerosis. This neuroprotection against oxidation conditions suggest that these metabolites could be interesting lead candidates in drug development for neurodegenerative diseases.

Full Text Available We present a novel non-invasive technique to measure the polarity of GABAergic responses based on cell-attached recordings of currents activated by laser-uncaging of GABA. For these recordings, a patch pipette was filled with a solution containing RuBi-GABA, and GABA was released from this complex by a laser beam conducted to the tip of the patch pipette via an optic fiber. In cell-attached recordings from neocortical and hippocampal neurons in postnatal days P2-5 rat brain slices in vitro, we found that laser-uncaging of GABA activates integral cell-attached currents mediated by tens of GABA(A channels. The initial response was inwardly directed, indicating a depolarizing response to GABA. The direction of the initial response was dependent on the pipette potential and analysis of its slope-voltage relationships revealed a depolarizing driving force of +11 mV for the currents through GABA channels. Initial depolarizing responses to GABA uncaging were inverted to hyperpolarizing in the presence of the NKCC1 blocker bumetanide. Current-voltage relationships of the currents evoked by Rubi-GABA uncaging using voltage-ramps at the peak of responses not only revealed a bumetanide-sensitive depolarizing reversal potential of the GABA(A receptor mediated responses, but also showed a strong voltage-dependent hysteresis. Upon desensitization of the uncaged-GABA response, current-voltage relationships of the currents through single GABA(A channels revealed depolarizing responses with the driving force values similar to those obtained for the initial response. Thus, cell-attached recordings of the responses evoked by local intrapipette GABA uncaging are suitable to assess the polarity of the GABA(A-Rs mediated signals in small cell compartments.

Oxygen deprivation triggers excitotoxic cell death in mammal neurons through excessive calcium loading via over-activation of N-methyl-d-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. This does not occur in the western painted turtle, which overwinters for months without oxygen. Neurological damage is avoided through anoxia-mediated decreases in NMDA and AMPA receptor currents that are dependent upon a modest rise in intracellular Ca(2+) concentrations ([Ca(2+)]i) originating from mitochondria. Anoxia also blocks mitochondrial reactive oxygen species (ROS) generation, which is another potential signaling mechanism to regulate glutamate receptors. To assess the effects of decreased intracellular [ROS] on NMDA and AMPA receptor currents, we scavenged ROS with N-2-mercaptopropionylglycine (MPG) or N-acetylcysteine (NAC). Unlike anoxia, ROS scavengers increased NMDA receptor whole-cell currents by 100%, while hydrogen peroxide decreased currents. AMPA receptor currents and [Ca(2+)]i concentrations were unaffected by ROS manipulation. Because decreases in [ROS] increased NMDA receptor currents, we next asked whether mitochondrial Ca(2+) release prevents receptor potentiation during anoxia. Normoxic activation of mitochondrial ATP-sensitive potassium (mKATP) channels with diazoxide decreased NMDA receptor currents and was unaffected by subsequent ROS scavenging. Diazoxide application following ROS scavenging did not rescue scavenger-mediated increases in NMDA receptor currents. Fluorescent measurement of [Ca(2+)]i and ROS levels demonstrated that [Ca(2+)]i increases before ROS decreases. We conclude that decreases in ROS concentration are not linked to anoxia-mediated decreases in NMDA/AMPA receptor currents but are rather associated with an increase in NMDA receptor currents that is prevented during anoxia by mitochondrial Ca(2+) release.

In the cerebral cortex, pyramidal cells and interneurons are generated in distant germinal zones, and so the mechanisms that control their precise assembly into specific microcircuits remain an enigma. Here we report that cortical interneurons labeled at the clonal level do not distribute randomly but rather have a strong tendency to cluster in the mouse neocortex. This behavior is common to different classes of interneurons, independently of their origin. Interneuron clusters are typically contained within one or two adjacentcortical layers, are largely formed by isochronically generated neurons and populate specific layers, as revealed by unbiased hierarchical clustering methods. Our results suggest that different progenitor cells give rise to interneurons populating infra- and supragranular cortical layers, which challenges current views of cortical neurogenesis. Thus, specific lineages of cortical interneurons seem to be produced to primarily mirror the laminar structure of the cerebral cortex, rather than its columnar organization.

Randomly-connected networks of integrate-and-fire (IF) neurons are known to display asynchronous irregular (AI) activity states, which resemble the discharge activity recorded in the cerebral cortex of awake animals. However, it is not clear whether such activity states are specific to simple IF models, or if they also exist in networks where neurons are endowed with complex intrinsic properties similar to electrophysiological measurements. Here, we investigate the occurrence of AI states in networks of nonlinear IF neurons, such as the adaptive exponential IF (Brette-Gerstner-Izhikevich) model. This model can display intrinsic properties such as low-threshold spike (LTS), regular spiking (RS) or fast-spiking (FS). We successively investigate the oscillatory and AI dynamics of thalamic, cortical and thalamocortical networks using such models. AI states can be found in each case, sometimes with surprisingly small network size of the order of a few tens of neurons. We show that the presence of LTS neurons in cortex or in thalamus, explains the robust emergence of AI states for relatively small network sizes. Finally, we investigate the role of spike-frequency adaptation (SFA). In cortical networks with strong SFA in RS cells, the AI state is transient, but when SFA is reduced, AI states can be self-sustained for long times. In thalamocortical networks, AI states are found when the cortex is itself in an AI state, but with strong SFA, the thalamocortical network displays Up and Down state transitions, similar to intracellular recordings during slow-wave sleep or anesthesia. Self-sustained Up and Down states could also be generated by two-layer cortical networks with LTS cells. These models suggest that intrinsic properties such as adaptation and low-threshold bursting activity are crucial for the genesis and control of AI states in thalamocortical networks.

Background and Purpose - Although the penumbra can be saved by early reperfusion, in the rat it is consistently affected by selective neuronal loss. Mapping selective neuronal loss in the living primate would be desirable. Methods - Five young adult baboons underwent {sup 15}O positron emission tomography for cerebral blood flow, cerebral oxygen consumption, and oxygen extraction fraction mapping at baseline and serially during and after 20-hours temporary middle cerebral artery occlusion. At approximately day 30, {sup 11}C-flumazenil (FMZ), a potential positron emission tomography marker of selective neuronal loss, and structural magnetic resonance-based infarct mapping were obtained, and the brain was perfused-fixed. Reduced FMZ binding in non-infarcted cortical middle cerebral artery areas was searched voxel-wise, and specific binding was assessed using compartmental modeling of FMZ time-activity curves. Results - Visual inspection revealed reduced late FMZ uptake in the affected cortical territory, extending well beyond the infarct. Accordingly, the incidence of selected voxels was greater than chance, documenting mildly but significantly reduced FMZ uptake and specific binding. Serial {sup 15}O positron emission tomography revealed moderately severe acute ischemia followed by reperfusion. Histopathology documented only mild neuronal changes in or near the affected areas. Conclusions - We document moderate but definite late FMZ binding decrements in non-infarcted cortical areas in the baboon, consistent with previous rat and human studies. These were acutely characterized by moderate ischemia followed by reperfusion, consistent with neuronal damage from ischemic or reperfusion injury in the salvaged at-risk tissue. Only mild histopathological changes subtended these FMZ alterations suggesting subtle processes such as isolated dendrite or synapse loss. Whether these changes impact on clinical outcome deserves studying because they may be targeted by specific

Maturation of electrical excitability during early postnatal development is critical to formation of functional neural circuitry in the mammalian neocortex. Little is known, however, about the changes in gene expression underlying the development of firing properties that characterize different classes of corticalneurons. Here we describe the development of corticalneurons with two distinct firing phenotypes, regular-spiking (RS) and fast-spiking (FS), that appear to emerge from a population of immature multiple-spiking (IMS) neurons during the first two postnatal weeks, both in vivo (within layer IV) and in vitro. We report the expression of a slowly inactivating, 4-AP-sensitive potassium current (K4-AP) at significantly higher density in FS compared with RS neurons. The same current is expressed at intermediate levels in IMS neurons. The kinetic, voltage-dependent, and pharmacological properties of the K4-AP current are similar to those observed by heterologous expression of Kv3.1 potassium channel mRNA. Single-cell RT-PCR analysis demonstrates that PCR products representing Kv3.1 transcripts are amplified more frequently from FS than RS neurons, with an intermediate frequency of Kv3.1 detection in neurons with immature firing properties. Taken together, these data suggest that the Kv3.1 gene encodes the K4-AP current and that expression of this gene is regulated in a cell-specific manner during development. Analysis of the effects of 4-AP on firing properties suggests that the K4-AP current is important for rapid action potential repolarization, fast after-hyperpolarization, brief refractory period, and high firing frequency characteristic of FS GABAergic interneurons.

Full Text Available Neuregulin receptor degradation protein-1 (Nrdp1 is an E3 ubiquitin ligase that targets proteins for degradation and regulates cell growth, apoptosis and oxidative stress in various cell types. We have previously shown that Nrdp1 is implicated in ischemic cardiomyocyte death. In this study, we investigated the change of Nrdp1 expression in ischemic neurons and its role in ischemic neuronal injury. Primary rat cerebral corticalneurons and pheochromocytoma (PC12 cells were infected with adenoviral constructs expressing Nrdp1 gene or its siRNA before exposing to oxygen-glucose deprivation (OGD treatment. Our data showed that Nrdp1 was upregulated in ischemic brain tissue 3 h after middle cerebral artery occlusion (MCAO and in OGD-treated neurons. Of note, Nrdp1 overexpression by Ad-Nrdp1 enhanced OGD-induced neuron apoptosis, while knockdown of Nrdp1 with siRNA attenuated this effect, implicating a role of Nrdp1 in ischemic neuron injury. Moreover, Nrdp1 upregulation is accompanied by increased protein ubiquitylation and decreased protein levels of ubiquitin-specific protease 8 (USP8 in OGD-treated neurons, which led to a suppressed interaction between USP8 and HIF-1α and subsequently a reduction in HIF-1α protein accumulation in neurons under OGD conditions. In conclusion, our data support an important role of Nrdp1 upregulation in ischemic neuronal death, and suppressing the interaction between USP8 and HIF-1α and consequently the hypoxic adaptive response of neurons may account for this detrimental effect.

Full Text Available Neurotrophin signaling is crucial for neuron growth. While the “signaling endosomes” hypothesis is one of the accepted models, the molecular machinery that drives retrograde axonal transport of TrkB signaling endosomes is largely unknown. In particular, mechanisms recruiting dynein to TrkB signaling endosomes have not been elucidated. Here, using snapin deficient mice and gene rescue experiments combined with compartmentalized cultures of live corticalneurons, we reveal that Snapin, as a dynein adaptor, mediates retrograde axonal transport of TrkB signaling endosomes. Such a role is essential for dendritic growth of corticalneurons. Deleting snapin or disrupting Snapin-dynein interaction abolishes TrkB retrograde transport, impairs BDNF-induced retrograde signaling from axonal terminals to the nucleus, and decreases dendritic growth. Such defects were rescued by reintroducing the snapin gene. Our study indicates that Snapin-dynein coupling is one of the primary mechanisms driving BDNF-TrkB retrograde transport, thus providing mechanistic insights into the regulation of neuronal growth and survival.

The differentiation of cortical interneurons is controlled by environmental factors. Here, we describe the role of activity and neurotrophins in regulating parvalbumin (PARV) expression using organotypic cultures (OTC) of rat visual cortex as model system. In OTC, PARV expression was dramatically delayed. The organotypic proportion of approximately 6% PARV neurons was not established before 50-70 DIV, whereas in vivo all neurons are present until P20. Thalamic afferents increased cortical PARV mRNA in OTC, but not to the age-matched in vivo level. During the first 10 DIV, BDNF and NT-4 accelerated PARV mRNA expression in a Trk receptor and MEK2 dependent manner. The BDNF action required PI3 kinase signalling. PARV expression required activity. The proportion of neurons which managed to up-regulate PARV was inversely related to the duration of early transient periods of activity deprivation. Long-term activity-deprived OTC completely failed to up-regulate PARV mRNA. Both TrkB ligands failed to promote PARV expression in activity-deprived OTC. However, a few basket and chandelier neurons were observed, suggesting that the development of class-specific morphological features is activity-independent. Once established, PARV expression became resistant to late-onset activity deprivation. In conclusion, PARV expression depended on activity and TrkB ligands which appear to prime the PARV expression already before its developmental onset.

Different mechanisms have been suggested to contribute to isoflurane-mediated neuroprotection. Previous studies have suggested that the protein Slit can abrogate neuronal death in mixed neuronal-glial cultures exposed to oxygen-glucose deprivation (OGD) and reperfusion (OGD/R). We hypothesized that isoflurane increases the expression of Slit and its receptor Robo when corticalneurons are exposed to OGD/R. To test this hypothesis, we exposed primary corticalneurons to OGD for 90 min and reperfusion for 24h and investigated how isoflurane post-conditioning affected cell survival and expression of Slit2 and receptors Robo1 and Robo4. Cell survival increased after administration of isoflurane, as assessed by the lactate dehydrogenase assay, trypan blue analysis, and propidium iodide staining. Western blot analysis showed that cleaved caspase-3 was increased after OGD/R(PSlit2 and Robo1, but not Robo4, were increased after OGD/R (PSlit2 and Robo1 expression. These findings provide a novel explanation for the pleiotropic effects of isoflurane that could benefit the central nervous system.

The present study established a rat corticalneuronal model of in vitro mechanical injury. At 30 min-utes after injury, the survival rate of the injured corticalneurons was decreased compared with normal neurons, and was gradually decreased with aggravated degree of injury. Reverse transcrip-tion-polymerase chain reaction results showed that at 1 hour after injury, there was increased ex-pression of metabotropic glutamate receptor 1a in corticalneurons. Immunohistochemical staining results showed that at 30 minutes after injury, the number of metabotropic glutamate receptor 1a-positive cells increased compared with normal neurons. At 12 hours after injury, lactate dehy-drogenase activity in the (RS)-1-aminoindan-1, 5-dicarboxylic acid (AIDA)-treated injury neurons was significantly decreased than that in the pure injury group. At 1 hour after injury, intracellular free Ca2+ concentration was markedly decreased in the AIDA-treated injury neurons than that in the pure injury neurons. These findings suggest that after mechanical injury to corticalneurons, metabotropic glutamate receptor 1a expression increased. The resulting increase in intracellular free Ca2+ con-centration was blocked by AIDA, indicating that AIDA exhibits neuroprotective effects after me-chanical injury.

The effects of S-alpha-amino-3-hydroxy-4-isoxozolepropionic acid (AMPA) lesions of the nucleus basalis magnocellularis on the M1/M2 nature of the responses of somatosensory corticalneurones to acetylcholine (ACh) in Sprague-Dawley rats were investigated by iontophoretic application and extracellular single unit recording. The responses were characterised using pirenzepine, an M1 receptor antagonist, and gallamine, an M2 antagonist. Eighty two neurones in control and 94 neurones in lesioned animals were studied. In control animals, 37% of responses to ACh were sensitive to pirenzepine, gallamine or to both antagonists. This increased to 62% in lesioned animals, the proportions of pirenzepine- and gallamine-sensitive responses remaining unchanged. These results provide the first electrophysiological confirmation that both pirenzepine- and gallamine-sensitive (M1 and M2) receptors occur postsynaptic to afferent cholinergic terminals and that their postsynaptic stimulation may produce both inhibition and excitation.

Previous studies using neural activity recording and neuroimaging techniques have reported functional deficits in the mirror neuron system (MNS) for individuals with autism spectrum disorder (ASD). However, a few studies focusing on gray and white matter structures of the MNS have yielded inconsistent results. The current study recruited adolescents and young adults with ASD (aged 15-26 years) and age-matched typically developing (TD) controls (aged 14-25 years). The cortical thickness (CT) and microstructural integrity of the tracts connecting the regions forming the classical MNS were investigated. High-resolution T1-weighted imaging and diffusion spectrum imaging were performed to quantify the CT and tract integrity, respectively. The structural covariance of the CT of the MNS regions revealed a weaker coordination of the MNS network in ASD. A strong correlation was found between the integrity of the right frontoparietal tracts and the social communication subscores measured by the Chinese version of the Social Communication Questionnaire. The results showed that there were no significant mean differences in the CTs and tract integrity between the ASD and TD groups, but revealed a moderate or even reverse age effect on the frontal MNS structures in ASD. In conclusion, aberrant structural coordination may be an underlying factor affecting the function of the MNS in ASD patients. The association between the right frontoparietal tracts and social communication performance implies a neural correlate of communication processing in the autistic brain. This study provides evidence of abnormal MNS structures and their influence on social communication in individuals with ASD.

Full Text Available Activity-dependent transcription of brain-derived neurotrophic factor (BDNF has been studied as an important model to elucidate the mechanisms underlying numerous aspects of neuroplasticity. It has been extensively emphasized that Ca(2+ influx through different routes may have significantly different effects on BDNF transcription. Here, we examined the regulatory property of the major calcium responsive elements (CaRE in BDNF promoter IV in cultured rat corticalneurons. BDNF promoter IV, as well as CaRE1 and CaRE3, was significantly activated by Ca(2+ influx through L-type voltage-gated calcium channel (L-VGCC or NMDA receptor (NMDAR. However, the L-VGCC- and NMDAR-mediated activation of CaRE was differentially regulated by different Ca(2+-stimulated protein kinases. Specifically, PKA, CaMKI, and CaMKIV activity were required for L-VGCC-, but not NMDAR-mediated CaRE1 activation. CaMKI activity was required for NMDAR- but not L-VGCC-mediated CaRE3 activation. Surprisingly, the activation of CaRF, a previously identified transcription factor for CaRE1, was stimulated via L-VGCC but not NMDAR, and required MEK, PI3K and CaMKII activity. These results suggest a new working model that activity-dependent BDNF IV up-regulation may be coordinately mediated by CaRE1 and CaRE3 activity, which show different responses to Ca(2+-stimulated kinases. Our data also explain how the individual cis-element in BDNF promoter is distinctively coupled to different Ca(2+ routes.

Full Text Available The neurons of the mammalian brain are generated by progenitors dividing either at the apical surface of the ventricular zone (neuroepithelial and radial glial cells, collectively referred to as apical progenitors or at its basal side (basal progenitors, also called intermediate progenitors. For apical progenitors, the orientation of the cleavage plane relative to their apical-basal axis is thought to be of critical importance for the fate of the daughter cells. For basal progenitors, the relationship between cell polarity, cleavage plane orientation and the fate of daughter cells is unknown. Here, we have investigated these issues at the very onset of cortical neurogenesis. To directly observe the generation of neurons from apical and basal progenitors, we established a novel transgenic mouse line in which membrane GFP is expressed from the beta-III-tubulin promoter, an early pan-neuronal marker, and crossed this line with a previously described knock-in line in which nuclear GFP is expressed from the Tis21 promoter, a pan-neurogenic progenitor marker. Mitotic Tis21-positive basal progenitors nearly always divided symmetrically, generating two neurons, but, in contrast to symmetrically dividing apical progenitors, lacked apical-basal polarity and showed a nearly randomized cleavage plane orientation. Moreover, the appearance of beta-III-tubulin-driven GFP fluorescence in basal progenitor-derived neurons, in contrast to that in apical progenitor-derived neurons, was so rapid that it suggested the initiation of the neuronal phenotype already in the progenitor. Our observations imply that (i the loss of apical-basal polarity restricts neuronal progenitors to the symmetric mode of cell division, and that (ii basal progenitors initiate the expression of neuronal phenotype already before mitosis, in contrast to apical progenitors.

of the negative shift in direct current potential by 33% (4.1 mV). Furthermore, the compound diminished the average depression of spontaneous electrocorticographic activity by 11% during first 40 min of post-cortical spreading depression recovery, but did not mitigate the suppressing effect of cortical spreading...... during the first hour after i.v. injection. The Tat-N-dimer suppressed stimulation-evoked synaptic activity by 2-20%, while cortical blood flow and cerebral oxygen metabolic (CMRO2) responses were preserved. During cortical spreading depression, the Tat-N-dimer reduced the average amplitude...

Mounting evidence suggests that the pathological hallmarks of Alzheimer's disease (AD) are caused by the intraneuronal accumulation of beta-amyloid protein (Aβ). Reuptake of extracellular Aβ is believed to contribute significantly to the intraneuronal Aβ pool in the early stages of AD. Published reports have claimed that the low-density lipoprotein receptor-related protein 1 (LRP1) mediates Aβ1-42 uptake and lysosomal trafficking in GT1-7 neuronal cells and mouse embryonic fibroblast non-neuronal cells. However, there is no direct evidence supporting the role of LRP1 in Aβ internalization in primary neurons. Our recent study indicated that p38 MAPK and ERK1/2 signaling pathways are involved in regulating α7 nicotinic acetylcholine receptor (α7nAChR)-mediated Aβ1-42 uptake in SH-SY5Y cells. This study was designed to explore the regulation of MAPK signaling pathways on LRP1-mediated Aβ internalization in neurons. We found that extracellular Aβ1-42 oligomers could be internalized into endosomes/lysosomes and mitochondria in corticalneurons. Aβ1-42 and LRP1 were also found co-localized in neurons during Aβ1-42 internalization, and they could form Aβ1-42-LRP1 complex. Knockdown of LRP1 expression significantly decreased neuronal Aβ1-42 internalization. Finally, we identified that p38 MAPK and ERK1/2 signaling pathways regulated the internalization of Aβ1-42 via LRP1. Therefore, these results demonstrated that LRP1, p38 MAPK and ERK1/2 mediated the internalization of Aβ1-42 in neurons and provided evidence that blockade of LRP1 or inhibitions of MAPK signaling pathways might be a potential approach to lowering brain Aβ levels and served a potential therapeutic target for AD.

Flavonoids from the stems and leaves of Scutellaria baicalensis Georgi, an antioxidant, marked-ly improve memory impairments and neuronal injuries. In the present study, primary corticalneurons of rats were exposed to potassium cyanide to establish a model of in vitro neural cell apoptosis. Inhibition of apoptosis by lfavonoids from the stems and leaves of Scutellaria baical-ensis Georgi at concentrations of 18.98, 37.36, and 75.92μg/mL was detected using this model. These lfavonoids dramatically increased cell survival, inhibited cell apoptosis and excessive pro-duction of malondialdehyde, and increased the activities of superoxide dismutase, glutathione peroxidase, and Na+-K+-ATPase in primary corticalneurons exposed to potassium cyanide. The lfavonoids from the stems and leaves of Scutellaria baicalensis Georgi were originally found to have a polyhydric structure and to protect against cerebral hypoxia in in vitro and in vivo models, including hypoxia induced by potassium cyanide or cerebral ischemia. The present study suggests that lfavonoids from the stems and leaves of Scutellaria baicalensis Georgi exert neuroprotective effects via modulation of oxidative stress, such as malondialdehyde, superoxide dismutase, gluta-thione peroxidase and Na+-K+-ATPase disorders induced by potassium cyanide.

Not only neuronal death but also neuritic atrophy and synaptic loss underlie the pathogenesis of Alzheimer's disease as direct causes of the memory deficit. Extracts of Siberian ginseng (the rhizome of Eleutherococcus senticosus) were shown to have protective effects on the regeneration of neurites and the reconstruction of synapses in rat cultured corticalneurons damaged by amyloid β (Aβ)(25-35), and eleutheroside B was one of the active constituents. In this study, a comprehensive evaluation of constituents was conducted to explore active components from Siberian ginseng which can protect against neuritic atrophy induced by Aβ(25-35) in cultured rat corticalneurons. The ethyl acetate, n-butanol and water fractions from the methanol extract of Siberian ginseng showed protective effects against Aβ-induced neuritic atrophy. Twelve compounds were isolated from the active fractions and identified. Among them, eleutheroside B, eleutheroside E and isofraxidin showed obvious protective effects against Aβ(25-35)-induced atrophies of axons and dendrites at 1 and 10 μM.

Our previous work has demonstrated that piracetam inhibited the decrease in amino acid content induced by chronic hypoperfusion, ameliorated the dysfunction of learning and memory in a hypoperfusion rat model, down-regulated P53, and BAX protein, facilitated the synaptic plasticity, and may be helpful in the treatment of vascular dementia. To explore the precise mechanism, the present study further evaluated effects of piracetam on Oxygen and glucose deprivation (OGD)-induced neuronal damage in rat primary cortical cells. The addition of piracetam to the cultured cells 12 h before OGD for 4 h significantly reduced neuronal damage as determined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and lactate dehydrogenase release experiments. Piracetam also lowered the levels of malondialdehyde, nitrogen monoxidum, and xanthine oxidase which was increased in the OGD cells, and enhanced the activities of superoxide dismutase and glutathione peroxidase, which were decreased in the OGD cells. We also demonstrated that piracetam could decrease glutamate and aspartate release when cortical cells were subjected to OGD. Furthermore, Western blot study demonstrated that piracetam attenuated the increased expression of P53 and BAX protein in OGD cells. These observations demonstrated that piracetam reduced OGD-induced neuronal damage by inhibiting the oxidative stress and decreasing excitatory amino acids release and lowering P53/Bax protein expression in OGD cells.

Measles virus (MV) infection may lead to severe chronic CNS disease processes, including MV-induced encephalitis. Because the intracellular Ca(2+) concentration ([Ca(2+)](i)) is a major determinant of the (patho-)physiological state in all cells we asked whether important Ca(2+) conducting pathways are affected by MV infection in cultured cortical rat neurons. Patch-clamp measurements revealed a decrease in voltage-gated Ca(2+) currents during MV-infection, while voltage-gated K(+) currents and NMDA-evoked currents were unaffected. Calcium-imaging experiments using 50mM extracellular KCl showed reduced [Ca(2+)](i) increases in MV-infected neurons, confirming a decreased activity of voltage-gated Ca(2+) channels. In contrast, the group-I metabotropic glutamate receptor (mGluR) agonist DHPG evoked changes in [Ca(2+)](i) that were increased in MV-infected cells. Our results show that MV infection conversely regulates Ca(2+) signals induced by group-I mGluRs and by voltage-gated Ca(2+) channels, suggesting that these physiological impairments may contribute to an altered function of corticalneurons during MV-induced encephalitis.

Autism spectrum disorders (ASDs) affect up to 1 in 68 children. Autism-specific autoantibodies directed against fetal brain proteins have been found exclusively in a subpopulation of mothers whose children were diagnosed with ASD or maternal autoantibody-related autism. We tested the impact of autoantibodies on brain development in mice by transferring human antigen-specific IgG directly into the cerebral ventricles of embryonic mice during cortical neurogenesis. We show that autoantibodies recognize radial glial cells during development. We also show that prenatal exposure to autism-specific maternal autoantibodies increased stem cell proliferation in the subventricular zone (SVZ) of the embryonic neocortex, increased adult brain size and weight, and increased the size of adult corticalneurons. We propose that prenatal exposure to autism-specific maternal autoantibodies directly affects radial glial cell development and presents a viable pathologic mechanism for the maternal autoantibody-related prenatal ASD risk factor.

Radiofrequency electromagnetic field (RF-EMF) is used globally in conjunction with mobile communications. There are public concerns of the perceived deleterious biological consequences of RF-EMF exposure. This study assessed neuronal effects of RF-EMF on the cerebral cortex of the mouse brain as a proxy for cranial exposure during mobile phone use. C57BL/6 mice were exposed to 835 MHz RF-EMF at a specific absorption rate (SAR) of 4.0 W/kg for 5 hours/day during 12 weeks. The aim was to examine activation of autophagy pathway in the cerebral cortex, a brain region that is located relatively externally. Induction of autophagy genes and production of proteins including LC3B-II and Beclin1 were increased and accumulation of autolysosome was observed in neuronal cell bodies. However, proapoptotic factor Bax was down-regulted in the cerebral cortex. Importantly, we found that RF-EMF exposure led to myelin sheath damage and mice displayed hyperactivity-like behaviour. The data suggest that autophagy may act as a protective pathway for the neuronal cell bodies in the cerebral cortex during radiofrequency exposure. The observations that neuronal cell bodies remained structurally stable but demyelination was induced in corticalneurons following prolonged RF-EMF suggests a potential cause of neurological or neurobehavioural disorders. PMID:28106136

Folate deficiency is accompanied by a decline in the cognitive neurotransmitter acetylcholine and a decline in cognitive performance in mice lacking apolipoprotein E (ApoE-/- mice), a low-density lipoprotein that regulates aspects of lipid metabolism. One direct consequence of folate deficiency is a decline in S-adenosylmethionine (SAM). Since dietary SAM supplementation maintains acetylcholine levels and cognitive performance in the absence of folate, we examined herein the impact of folate and SAM on neuronal synaptic activity. Embryonic corticalneurons from mice expressing or lacking ApoE (ApoE+/+ or -/-, respectively) were cultured for 1 month on multi-electrode arrays, and signaling was recorded. ApoE+/+ cultures displayed significantly more frequent spontaneous signals than ApoE-/- cultures. Supplementation with 166 µm SAM (not normally present in culture medium) increased signal frequency and decreased signal amplitude in ApoE+/+ cultures. SAM also increased the frequency of tightly clustered signal bursts. Folate deprivation reversibly reduced signal frequency in ApoE+/+ cultures; SAM supplementation maintained signal frequency despite folate deprivation. These findings support the importance of dietary supplementation with folate and SAM on neuronal health. Supplementation with 166 µm SAM did not alter signaling in ApoE-/- cultures, which may be a reflection of the reduced SAM levels in ApoE-/- mice. The differential impact of SAM on ApoE+/+ and -/- neurons underscores the combined impact of nutritional and genetic deficiencies on neuronal homeostasis.

Many different types of integrate-and-fire models have been designed in order to explain how it is possible for a corticalneuron to integrate over many independent inputs while still producing highly variable spike trains. Within this context, the variability of spike trains has been almost exclusively measured using the coefficient of variation of interspike intervals. However, another important statistical property that has been found in cortical spike trains and is closely associated with their high firing variability is long-range dependence. We investigate the conditions, if any, under which such models produce output spike trains with both interspike-interval variability and long-range dependence similar to those that have previously been measured from actual corticalneurons. We first show analytically that a large class of high-variability integrate-and-fire models is incapable of producing such outputs based on the fact that their output spike trains are always mathematically equivalent to renewal processes. This class of models subsumes a majority of previously published models, including those that use excitation-inhibition balance, correlated inputs, partial reset, or nonlinear leakage to produce outputs with high variability. Next, we study integrate-and-fire models that have (nonPoissonian) renewal point process inputs instead of the Poisson point process inputs used in the preceding class of models. The confluence of our analytical and simulation results implies that the renewal-input model is capable of producing high variability and long-range dependence comparable to that seen in spike trains recorded from corticalneurons, but only if the interspike intervals of the inputs have infinite variance, a physiologically unrealistic condition. Finally, we suggest a new integrate-and-fire model that does not suffer any of the previously mentioned shortcomings. By analyzing simulation results for this model, we show that it is capable of producing output

Full Text Available Introduction and Objectives: Selective neuronal loss (SNL in the reperfused penumbra may impact clinical recovery and is thus important to investigate. Brief proximal middle cerebral artery occlusion (MCAo results in predominantly striatal SNL, yet cortical damage is more relevant given its behavioral implications and that thrombolytic therapy mainly rescues the cortex. Distal temporary MCAo (tMCAo does target the cortex, but the optimal occlusion duration that results in isolated SNL has not been determined. In the present study we assessed different distal tMCAo durations looking for consistently pure SNL.Methods: Microclip distal tMCAo (md-tMCAo was performed in ~6-month old male spontaneously hypertensive rats (SHRs. We previously reported that 45min md-tMCAo in SHRs results in pan-necrosis in the majority of subjects. Accordingly, three shorter MCAo durations were investigated here in decremental succession, namely 30, 22 and 15mins (n=3, 3 and 7 subjects, respectively. Recanalization was confirmed by MR angiography just prior to brain collection at 28 days and T2-weighted MRI was obtained for characterization of ischemic lesions. NeuN, OX42 and GFAP immunohistochemistry appraised changes in neurons, microglia and astrocytes, respectively. Ischemic lesions were categorized into three main types: 1 pan-necrosis; 2 partial infarction; and 3 SNL. Results: Pan-necrosis or partial infarction was present in all 30min and 22min subjects, but not in the 15min group (p < 0.001, in which isolated cortical SNL was consistently present. MRI revealed characteristic hyperintense abnormalities in all rats with pan-necrosis or partial infarction, but no change in any 15min subject. Conclusions: We found that 15min distal MCAo consistently resulted in pure cortical SNL, whereas durations equal or longer than 22min consistently resulted in infarcts. This model may be of use to study the pathophysiology of cortical SNL and its prevention by appropriate

Full Text Available Ground squirrel, a hibernating mammalian species, is more resistant to ischemic brain stress than rat. Gaining insight into the adaptive mechanisms of ground squirrels may help us design treatment strategies to reduce brain damage in patients suffering ischemic stroke. To understand the anti-stress mechanisms in ground squirrel neurons, we studied glutamate toxicity in primary cultured neurons of the Daurian ground squirrel (Spermophilus dauricus. At the neuronal level, for the first time, we found that ground squirrel was more resistant to glutamate excitotoxicity than rat. Mechanistically, ground squirrel neurons displayed a similar calcium influx to the rat neurons in response to glutamate or N-methyl-D-aspartate (NMDA perfusion. However, the rate of calcium removal in ground squirrel neurons was markedly faster than in rat neurons. This allows ground squirrel neurons to maintain lower level of intracellular calcium concentration ([Ca2+]i upon glutamate insult. Moreover, we found that Na+/Ca2+ exchanger (NCX activity was higher in ground squirrel neurons than in rat neurons. We also proved that overexpression of ground squirrel NCX2, rather than NCX1 or NCX3, in rat neurons promoted neuron survival against glutamate toxicity. Taken together, our results indicate that ground squirrel neurons are better at maintaining calcium homeostasis than rat neurons and this is likely achieved through the activity of ground squirrel NCX2. Our findings not only reveal an adaptive mechanism of mammalian hibernators at the cellular level, but also suggest that NCX2 of ground squirrel may have therapeutic value for suppressing brain ischemic damage.

Ground squirrel, a hibernating mammalian species, is more resistant to ischemic brain stress than rat. Gaining insight into the adaptive mechanisms of ground squirrels may help us design treatment strategies to reduce brain damage in patients suffering ischemic stroke. To understand the anti-stress mechanisms in ground squirrel neurons, we studied glutamate toxicity in primary cultured neurons of the Daurian ground squirrel (Spermophilus dauricus). At the neuronal level, for the first time, we found that ground squirrel was more resistant to glutamate excitotoxicity than rat. Mechanistically, ground squirrel neurons displayed a similar calcium influx to the rat neurons in response to glutamate or N-methyl-D-aspartate (NMDA) perfusion. However, the rate of calcium removal in ground squirrel neurons was markedly faster than in rat neurons. This allows ground squirrel neurons to maintain lower level of intracellular calcium concentration ([Ca2+]i) upon glutamate insult. Moreover, we found that Na+/Ca2+ exchanger (NCX) activity was higher in ground squirrel neurons than in rat neurons. We also proved that overexpression of ground squirrel NCX2, rather than NCX1 or NCX3, in rat neurons promoted neuron survival against glutamate toxicity. Taken together, our results indicate that ground squirrel neurons are better at maintaining calcium homeostasis than rat neurons and this is likely achieved through the activity of ground squirrel NCX2. Our findings not only reveal an adaptive mechanism of mammalian hibernators at the cellular level, but also suggest that NCX2 of ground squirrel may have therapeutic value for suppressing brain ischemic damage. PMID:25415196

, following light absorption of a photosensitizer (PS) at specific subcellular localizations. Here we use a hydrophilic PS based on a palladium porphyrin protected with a dendritic structure, rendering it impermeable to the cell membrane. Whole-cell electrophysiological recordings in HEPES-buffered medium...... (ABM) were made from cultured rat corticalneurons to provide insight into the events following extracellular generation of 1O2. Membrane resistance (Rm), capacitance (Cm), holding current (Ihold), and firing properties were monitored throughout. The V/I relationship was investigated with 1 s duration...... after washing, showing that PS had not associated with the neuron. Both controls: a) application of ABM alone with high irradiance (~7 J/cm2); b) application of PS without light, did not alter the investigated properties. The application of light in the presence of the PS affected the passive properties...

Full Text Available Rett Syndrome (RTT is a neurodevelopmental disorder predominantly caused by mutations in the X-linked gene MECP2. A primary feature of the syndrome is the impaired maturation and maintenance of excitatory synapses in the central nervous system (CNS. Different RTT mouse models have shown that particular Mecp2 mutations have highly variable effects on neuronal architecture. Distinguishing MeCP2 mutant cellular phenotypes therefore demands analysis of specific mutations in well-defined neuronal subpopulations. We examined a transgenically labeled subset of corticalneurons in YFP-H mice crossed with the Mecp2(tm1.1Jae mutant line. YFP(+ Layer 5 pyramidal neurons in the motor cortex of wildtype and hemizygous mutant male mice were examined for differences in dendrite morphology and spine density. Total basal dendritic length was decreased by 18.6% due to both shorter dendrites and reduced branching proximal to the soma. Tangential dendrite lengths in the apical tuft were reduced by up to 26.6%. Spine density was reduced by 47.4% in the apical tuft and 54.5% in secondary apical dendrites, but remained unaffected in primary apical and proximal basal dendrites. We also found that MeCP2 mutation reduced the number of YFP(+ cells in YFP-H mice by up to 72% in various cortical regions without affecting the intensity of YFP expression in individual cells. Our results support the view that the effects of MeCP2 mutation are highly context-dependent and cannot be generalized across mutation types and cell populations.

Full Text Available We identified a novel neuroprotective compound, 1-methoxyoctadecan-1-ol, from Uncaria sinensis (Oliv. Havil and investigated its effects and mechanisms in primary corticalneurons and in a photothrombotic ischemic model. In primary rat corticalneurons against glutamate-induced neurotoxicity, pretreatment with 1-methoxyoctadecan-1-ol resulted in significantly reduced neuronal death in a dose-dependent manner. In addition, treatment with 1-methoxyoctadecan-1-ol resulted in decreased neuronal apoptotic death, as assessed by nuclear morphological approaches. To clarify the neuroprotective mechanism of 1-methoxyoctadecan-1-ol, we explored the downstream signaling pathways of N-methyl-D-aspartate receptor (NMDAR with calpain activation. Treatment with glutamate leads to early activation of NMDAR, which in turn leads to calpain-mediated cleavage of striatal-enriched protein tyrosine phosphatase (STEP and subsequent activation of p38 mitogen activated protein kinase (MAPK. However, pretreatment with 1-methoxyoctadecan-1-ol resulted in significantly attenuated activation of GluN2B-NMDAR and a decrease in calpain-mediated STEP cleavage, leading to subsequent attenuation of p38 MAPK activation. We confirmed the critical role of p38 MAPK in neuroprotective effects of 1-methoxyoctadecan-1-ol using specific inhibitor SB203580. In the photothrombotic ischemic injury in mice, treatment with 1-methoxyoctadecan-1-ol resulted in significantly reduced infarct volume, edema size, and improved neurological function. 1-methoxyoctadecan-1-ol effectively prevents cerebral ischemic damage through down-regulation of calpain-mediated STEP cleavage and activation of p38 MAPK. These results suggest that 1-methoxyoctadecan-1-ol showed neuroprotective effects through down-regulation of calpain-mediated STEP cleavage with activation of GluN2B-NMDAR, and subsequent alleviation of p38 MAPK activation. In addition, 1-methoxyoctadecan-1-ol might be a useful therapeutic agent for

Full Text Available Acute brain injury is an important health problem. Given the critical position of caspase 8 at the crossroads of cell death pathways, we generated a new viable mouse line (Ncasp8(-/-, in which the gene encoding caspase 8 was selectively deleted in neurons by cre-lox system.Caspase 8 deletion reduced rates of neuronal cell death in primary neuronal cultures and in whole brain organotypic coronal slice cultures prepared from 4 and 8 month old mice and cultivated up to 14 days in vitro. Treatments of cultures with recombinant murine TNFα (100 ng/ml or TRAIL (250 ng/mL plus cyclohexamide significantly protected neurons against cell death induced by these apoptosis-inducing ligands. A protective role of caspase 8 deletion in vivo was also demonstrated using a controlled cortical impact (CCI model of traumatic brain injury (TBI and seizure-induced brain injury caused by kainic acid (KA. Morphometric analyses were performed using digital imaging in conjunction with image analysis algorithms. By employing virtual images of hundreds of brain sections, we were able to perform quantitative morphometry of histological and immunohistochemical staining data in an unbiased manner. In the TBI model, homozygous deletion of caspase 8 resulted in reduced lesion volumes, improved post-injury motor performance, superior learning and memory retention, decreased apoptosis, diminished proteolytic processing of caspases and caspase substrates, and less neuronal degeneration, compared to wild type, homozygous cre, and caspase 8-floxed control mice. In the KA model, Ncasp8(-/- mice demonstrated superior survival, reduced seizure severity, less apoptosis, and reduced caspase 3 processing. Uninjured aged knockout mice showed improved learning and memory, implicating a possible role for caspase 8 in cognitive decline with aging.Neuron-specific deletion of caspase 8 reduces brain damage and improves post-traumatic functional outcomes, suggesting an important role for this

pathways in neurons derived from human induced pluripotent stem cells (hiPSC). With this aim, cultures of hiPSC-derived neurons were incubated with [U-(13)C]glucose, [U-(13)C]glutamate or [U-(13)C]glutamine. Isotopic labeling in metabolites was determined using gas chromatography coupled to mass...... acid (TCA) cycle. This finding is supported by the extracellular acidification and oxygen consumption rates measured in the cultured human neurons. [U-(13)C]Glutamate and [U-(13)C]glutamine were found to be efficient energy substrates for the neuronal cultures originating from both mice and humans...

The constitutive and activity-dependent components of protein synthesis are both critical for neural function. Although the mechanisms controlling extracellularly induced protein synthesis are becoming clear, less is understood about the molecular networks that regulate the basal translation rate. Here we describe the effects of chronic treatment with various neurotrophic factors and cytokines on the basal rate of protein synthesis in primary corticalneurons. Among the examined factors, brain-derived neurotrophic factor (BDNF) showed the strongest effect. The rate of protein synthesis increased in the cortical tissues of BDNF transgenic mice, whereas it decreased in BDNF knock-out mice. BDNF specifically increased the level of the active, unphosphorylated form of eukaryotic elongation factor 2 (eEF2). The levels of active eEF2 increased and decreased in BDNF transgenic and BDNF knock-out mice, respectively. BDNF decreased kinase activity and increased phosphatase activity against eEF2 in vitro. Additionally, BDNF shortened the ribosomal transit time, an index of translation elongation. In agreement with these results, overexpression of eEF2 enhanced protein synthesis. Taken together, our results demonstrate that the increased level of active eEF2 induced by chronic BDNF stimulation enhances translational elongation processes and increases the total rate of protein synthesis in neurons.

Spike-wave discharges are electroencephalographic hallmarks of absence epilepsy. Spike-wave discharges are known to originate from thalamo-corticalneuronal network that normally produces sleep spindle oscillations. Although both sleep spindles and spike-wave discharges are considered as thalamo-cortical oscillations, functional relationship between them is still uncertain. The present study describes temporal dynamics of spike-wave discharges and sleep spindles as determined in long-time electroencephalograms (EEG) recorded in WAG/Rij rat model of absence epilepsy. We have proposed the wavelet-based method for the automatic detection of spike-wave discharges, sleep spindles (10-15Hz) and 5-9Hz oscillations in EEG. It was found that non-linear dynamics of spike-wave discharges and sleep spindles fits well to the law of 'on-off intermittency'. Intermittency in sleep spindles and spike-wave discharges implies that (1) temporal dynamics of these oscillations are deterministic in nature, and (2) it might be controlled by a system-level mechanism responsible for circadian modulation of neuronal network activity.

Full Text Available Abstract Background Apoptosis plays a key role in cell death observed in neurodegenerative diseases marked by a progressive loss of neurons as seen in Alzheimer's disease. Although the exact cause of apoptosis is not known, a number of factors such as free radicals, insufficient levels of nerve growth factors and excessive levels of glutamate have been implicated. We and others, have previously reported that in a stable HT22 neuronal cell line, glutamate induces apoptosis as indicated by DNA fragmentation and up- and down-regulation of Bax (pro-apoptotic, and Bcl-2 (anti-apoptotic genes respectively. Furthermore, these changes were reversed/inhibited by estrogens. Several lines of evidence also indicate that a family of cysteine proteases (caspases appear to play a critical role in neuronal apoptosis. The purpose of the present study is to determine in primary cultures of cortical cells, if glutamate-induced neuronal apoptosis and its inhibition by estrogens involve changes in caspase-3 protease and whether this process is mediated by Fas receptor and/or mitochondrial signal transduction pathways involving release of cytochrome c. Results In primary cultures of rat cortical cells, glutamate induced apoptosis that was associated with enhanced DNA fragmentation, morphological changes, and up-regulation of pro-caspase-3. Exposure of cortical cells to glutamate resulted in a time-dependent cell death and an increase in caspase-3 protein levels. Although the increase in caspase-3 levels was evident after 3 h, cell death was only significantly increased after 6 h. Treatment of cells for 6 h with 1 to 20 mM glutamate resulted in a 35 to 45% cell death that was associated with a 45 to 65% increase in the expression of caspase-3 protein. Pretreatment with caspase-3-protease inhibitor z-DEVD or pan-caspase inhibitor z-VAD significantly decreased glutamate-induced cell death of cortical cells. Exposure of cells to glutamate for 6 h in the presence or

BACKGROUND: Coriaria lactone-activated astrocytes released bioactive substances that eventually caused epilepsy.OBJECTIVE: It has been suggested that activated astrocytes alter the expression of the estrogen receptor and progesterone receptor by releasing bioactive substances during epilepsy, thereby affecting neuronal activity in the brain. This study was designed to observe the expression of the estrogen receptor and the progesterone receptor in rat brain following lateral ventricle injection of coriaria lactone-activated, astrocyte-conditioned medium.DESIGN AND SETTING: This immunohistochemical, randomized, controlled, animal study was conducted at the Department of Pathology, Hospital Affiliated to Binzhou Medical College, China.MATERIAL: Coriaria lactone was provided by Huaxi Pharmaceutical Factory, China.METHODS: Forty adult, healthy, male, Sprague Dawley rats were randomly assigned into two groups. Astrocyte-conditioned medium (10 μL) was injected into rat lateral ventricle in the control group (n = 8). Coriaria lactone-activated, astrocyte-conditioned medium (10 μL) was infused into the rat lateral ventricle in the coriaria lactone group (n = 32). At 2, 4, 8 and 12 hours following injection, rats were sacrificed and subjected to immunohistochemistry. Eight rats were studied at each time point.MAIN OUTCOME MEASURES: Behavioral changes were observed in rats of both groups. Expression of the estrogen receptor and the progesterone receptor in rat cortical and hippocampal neurons was measured using immunohistochemistry.RESULTS: Four hours after injection, estrogen receptor levels in rat cortical and hippocampal neurons were significantly higher in the coriaria lactone group than in the control group (P < 0.05). Progesterone receptor levels were significantly lower in the coriaria lactone group than in the control group (P < 0.05). Seizures were not observed in the control group. In the coriaria lactone group, convulsions appeared 30 minutes after injection

The present study has been designed to explore the molecular mechanism and signaling pathway targets of chlorogenic acid (CGA) and its main hydrolysates, caffeic (CA) and quinic acid in the protective effect against glutamate-excitotoxicity. For this purpose 8-DIV corticalneurons in primary culture were exposed to 50 μM L-glutamic acid plus 10 µM glycine, with or without 10-100 μM tested compounds. Chlorogenic acid and caffeic acid via their antioxidant properties inhibited cell death induced by glutamate in dose depended manner. However, quinic acid slightly protects neurons at a higher dose. DCF, JC-1 and Ca(2+)sensitive fluorescent dye fura-2, were used to measure intracellular ROS accumulation, mitochondrial membrane potential integration and intracellular calcium concentration [Ca(2+)] i . Results indicate that similarly, CGA acts as a protective agent against glutamate-induced corticalneurons injury by suppressing the accumulation of endogenous ROS and restore the mitochondrial membrane potential, activate the enzymatic antioxidant system by the increase levels of SOD activity and modulate the rise of intracellular calcium levels by increasing the rise of intracellular concentrations of Ca(2+)caused by glutamate overstimulation. PKC signaling cascade appear to be engaged in this protective mechanism. Interseling, CGA and CA also exhibit antiapoptotic properties against glutamate-induced cleaved activation of pro-caspases; caspase 1,8 and 9 and calpain (PD 150606,Calpeptin and MDL 28170).These data suggest that neuroprotective activity of CGA ester may occurs throught its hydrolysate,the caffeic acid and its interaction with intracellular molecules suggesting that CGA exert its neuroprotection via its caffeoly acid group that might potentially be used as a therapeutic agent in neurodegeneratives disorders associated with glutamate excitotoxicity.

Although recent reports have suggested that synchronous neuronal UP states are mediated by astrocytic activity, the mechanism responsible for this remains unknown. Astrocytic glutamate release synchronously depolarizes adjacentneurons, while synaptic transmissions are blocked. The purpose of this study was to confirm that astrocytic depolarization, propagated through synaptic connections, can lead to synchronous neuronal UP states. We applied astrocytic currents to local neurons in a neural network consisting of model corticalneurons. Our results show that astrocytic depolarization may generate synchronous UP states for hundreds of milliseconds in neurons even if they do not directly receive glutamate release from the activated astrocyte.

Full Text Available During embryonic development the preoptic area (POA gives rise to two populations of neurons which are generated at the same time, cortical interneurons and striatal cells. POA-derived cortical interneurons take a superficial path and avoid the developing striatum when they migrate to their target region. We found that EphB1, which is expressed in the striatal anlage, prevents cortical interneurons from entering the striatum via ephrin-B3 reverse signaling. In contrast, for striatal neurons which also express ephrin-B3, EphB1 acts as a stop signal. This dual role of EphB1 is due to differences in ephrin-B3 reverse signaling cascades. For striatal neurons, binding of EphB1 to ephrin-B3 reduces endogenously high levels of pSrc and pFAK, which then causes the cells to stop migration. In contrast, in cortical interneurons EphB1-ephrin-B3 reverse signaling leads to phosphorylation of Src and FAK which then mediates repulsion. Consistent with these in vitro findings, in an ephrin-B3 knockout mouse line, we discovered misrouted cortical interneurons in the striatum and an over-migration of striatal neurons in their target region. Thus, EphB1/ephrin-B3 reverse signaling has a different impact on two sets of neurons which are generated at the same time and place: it can act as a repulsive cue for migrating neurons or it can terminate neuronal migration, a novel role of the Eph/ephrin system.

The goal of the present study was to maximally alleviate the negative impact of stroke by increasing the therapeutic potency of injected mesenchymal multipotent stromal cells (MMSCs). To pursue this goal, the intercellular communications of MMSCs and neuronal cells were studied in vitro. As a result of cocultivation of MMSCs and rat corticalneurons, we proved the existence of intercellular contacts providing transfer of cellular contents from one cell to another. We present evidence of intercellular exchange with fluorescent probes specifically occupied by cytosol with preferential transfer from neurons toward MMSCs. In contrast, we observed a reversed transfer of mitochondria (from MMSCs to neural cells). Intravenous injection of MMSCs in a postischemic period alleviated the pathological indexes of a stroke, expressed as a lower infarct volume in the brain and partial restoration of neurological status. Also, MMSCs after cocultivation with neurons demonstrated more profound neuroprotective effects than did unprimed MMSCs. The production of the brain-derived neurotrophic factor was slightly increased in MMSCs, and the factor itself was redistributed in these cells after cocultivation. The level of Miro1 responsible for intercellular traffic of mitochondria was increased in MMSCs after cocultivation. We conclude that the exchange by cellular compartments between neural and stem cells improves MMSCs' protective abilities for better rehabilitation after stroke. This could be used as an approach to enhance the therapeutic benefits of stem cell therapy to the damaged brain. The idea of priming stem cells before practical use for clinical purposes was applied. Thus, cells were preconditioned by coculturing them with the targeted cells (i.e., neurons for the treatment of brain pathological features) before the transfusion of stem cells to the organism. Such priming improved the capacity of stem cells to treat stroke. Some additional minimal study will be required to

Full Text Available Down Syndrome (DS is a highly prevalent developmental disorder, affecting 1/700 births. Intellectual disability, which affects learning and memory, is present in all cases and is reflected by below average IQ. We sought to determine whether defective morphology and connectivity in neurons of the cerebral cortex may underlie the cognitive deficits that have been described in two mouse models of DS, the Tc1 and Ts1Rhr mouse lines. We utilised in utero electroporation to label a cohort of future upper layer projection neurons in the cerebral cortex of developing mouse embryos with GFP, and then examined neuronal positioning and morphology in early adulthood, which revealed no alterations in cortical layer position or morphology in either Tc1 or Ts1Rhr mouse cortex. The number of dendrites, as well as dendrite length and branching was normal in both DS models, compared with wildtype controls. The sites of projection neuron synaptic inputs, dendritic spines, were analysed in Tc1 and Ts1Rhr cortex at three weeks and three months after birth, and significant changes in spine morphology were observed in both mouse lines. Ts1Rhr mice had significantly fewer thin spines at three weeks of age. At three months of age Tc1 mice had significantly fewer mushroom spines--the morphology associated with established synaptic inputs and learning and memory. The decrease in mushroom spines was accompanied by a significant increase in the number of stubby spines. This data suggests that dendritic spine abnormalities may be a more important contributor to cognitive deficits in DS models, rather than overall neuronal architecture defects.

Neuronal respiration is controlled by ATP demand and Ca2+ but the roles played by each are unknown, as any Ca2+ signal also impacts on ATP demand. Ca2+ can control mitochondrial function through Ca2+-regulated mitochondrial carriers, the aspartate-glutamate and ATP-Mg/Pi carriers, ARALAR/AGC1 and SCaMC-3, respectively, or in the matrix after Ca2+ transport through the Ca2+ uniporter. We have studied the role of Ca2+ signaling in the regulation of mitochondrial respiration in intact mouse corticalneurons in basal conditions and in response to increased workload caused by increases in [Na+]cyt (veratridine, high-K+ depolarization) and/or [Ca2+]cyt (carbachol). Respiration in nonstimulated neurons on 2.5-5 mm glucose depends on ARALAR-malate aspartate shuttle (MAS), with a 46% drop in aralar KO neurons. All stimulation conditions induced increased OCR (oxygen consumption rate) in the presence of Ca2+, which was prevented by BAPTA-AM loading (to preserve the workload), or in Ca2+-free medium (which also lowers cell workload). SCaMC-3 limits respiration only in response to high workloads and robust Ca2+ signals. In every condition tested Ca2+ activation of ARALAR-MAS was required to fully stimulate coupled respiration by promoting pyruvate entry into mitochondria. In aralar KO neurons, respiration was stimulated by veratridine, but not by KCl or carbachol, indicating that the Ca2+ uniporter pathway played a role in the first, but not in the second condition, even though KCl caused an increase in [Ca2+]mit. The results suggest a requirement for ARALAR-MAS in priming pyruvate entry in mitochondria as a step needed to activate respiration by Ca2+ in response to moderate workloads.

Dopamine D4 receptor (D4R), which is strongly linked to neuropsychiatric disorders, such as attention-deficit hyperactivity disorder and schizophrenia, is highly expressed in pyramidal neurons and GABAergic interneurons in prefrontal cortex (PFC). In this study, we examined the impact of D4R on the excitability of these 2 neuronal populations. We found that D4R activation decreased the frequency of spontaneous action potentials (sAPs) in PFC pyramidal neurons, whereas it induced a transient increase followed by a decrease of sAP frequency in PFC parvalbumin-positive (PV+) interneurons. D4R activation also induced distinct effects in both types of PFC neurons on spontaneous excitatory and inhibitory postsynaptic currents, which drive the generation of sAP. Moreover, dopamine substantially decreased sAP frequency in PFC pyramidal neurons, but markedly increased sAP frequency in PV+ interneurons, and both effects were partially mediated by D4R activation. In the phencyclidine model of schizophrenia, the decreasing effect of D4R on sAP frequency in both types of PFC neurons was attenuated, whereas the increasing effect of D4R on sAP in PV+ interneurons was intact. These results suggest that D4R activation elicits distinct effects on synaptically driven excitability in PFC projection neurons versus fast-spiking interneurons, which are differentially altered in neuropsychiatric disorder-related conditions.

Full Text Available Using paired-end RNA sequencing, we have quantified the deep transcriptional changes that occur during differentiation of murine embryonic stem cells into a highly enriched population of glutamatergic corticalneurons. These data provide a detailed and nuanced account of longitudinal changes in the transcriptome during neurogenesis and neuronal maturation, starting from mouse embryonic stem cells and progressing through neuroepithelial stem cell induction, radial glial cell formation, neurogenesis, neuronal maturation and cortical patterning. Understanding the transcriptional mechanisms underlying the differentiation of stem cells into mature, glutamatergic neurons of cortical identity has myriad applications, including the elucidation of mechanisms of cortical patterning; identification of neurogenic processes; modeling of disease states; detailing of the host cell response to neurotoxic stimuli; and determination of potential therapeutic targets. In future work we anticipate correlating changes in longitudinal gene expression to other cell parameters, including neuronal function as well as characterizations of the proteome and metabolome. In this data article, we describe the methods used to produce the data and present the raw sequence read data in FASTQ files, sequencing run statistics and a summary flatfile of raw counts for 22,164 genes across 31 samples, representing 3-5 biological replicates at each timepoint. We propose that this data will be a valuable contribution to diverse research efforts in bioinformatics, stem cell research and developmental neuroscience studies.

Glutamate-induced excitotoxicity is involved in many neurological diseases. Preso, a novel postsynaptic scaffold protein, mediates excitatory synaptic transmission and various synaptic functions. In this study, we investigated the role of Preso in the regulation of glutamate-induced excitotoxicity in rat corticalneurons. Knockdown of Preso with small interfering RNA improved neuronal viability and attenuated the elevation of lactate dehydrogenase (LDH) release after glutamate treatment. Downregulation of Preso also inhibited an increase in the BAX/Bcl-2 ratio and cleavage of caspase-9 and caspase-3. Although the expression and distribution of metabotropic glutamate receptor (mGluR) 1/5, NR1, NR2A and NR2B were not changed by knockdown of Preso, downregulation of Preso protected neurons from glutamate-induced excitotoxicity by inhibiting mGluR and N-methyl-D-aspartate receptor function. However, downregulation of Preso neither affected the expression of GluR1 and GluR2 nor influenced the function of α-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor after glutamate treatment. Furthermore, intracellular Ca(2+) was an important downstream effector of Preso in the regulation of excitotoxicity. These results suggest that expression of Preso promotes the induction of excitotoxicity by facilitating different glutamate receptor signaling pathways. Therefore, Preso might be a potential pharmacological target for preventing and treating neurological diseases.

Low birth weight due to intrauterine growth retardation (IUGR) is suggested to be a risk factor for various psychiatric disorders such as schizophrenia. It has been reported that developmental cortical dysfunction and neurocognitive deficits are observed in individuals with IUGR, however, the underlying molecular mechanisms have yet to be elucidated. Brain-derived neurotrophic factor (BDNF) and its receptor TrkB are associated with schizophrenia and play a role in cortical development. We previously demonstrated that BDNF induced glutamate release through activation of the TrkB/phospholipase C-γ (PLC-γ) pathway in developing cultured corticalneurons, and that, using a rat model for IUGR caused by maternal administration of thromboxane A2, cortical levels of TrkB were significantly reduced in IUGR rats at birth. These studies prompted us to hypothesize that TrkB reduction in IUGR cortex led to impairment of BDNF-dependent glutamatergic neurotransmission. In the present study, we found that BDNF-induced glutamate release was strongly impaired in cultured IUGR corticalneurons where TrkB reduction was maintained. Impairment of BDNF-induced glutamate release in IUGR neurons was ameliorated by transfection of human TrkB (hTrkB). Although BDNF-stimulated phosphorylation of TrkB and of PLC-γ was decreased in IUGR neurons, the hTrkB transfection recovered the deficits in their phosphorylation. These results suggest that TrkB reduction causes impairment of BDNF-stimulated glutamatergic function via suppression of TrkB/PLC-γ activation in IUGR corticalneurons. Our findings provide molecular insights into how IUGR links to downregulation of BDNF function in the cortex, which might be involved in the development of IUGR-related diseases such as schizophrenia.

Full Text Available Abstract Background Brain-derived neurotrophic factor (BDNF, which is sorted into a regulated secretory pathway of neurons, is supposed to act retrogradely through dendrites on presynaptic neurons or anterogradely through axons on postsynaptic neurons. Depending on which is the case, the pattern and direction of trafficking of BDNF in dendrites and axons are expected to be different. To address this issue, we analyzed movements of green fluorescent protein (GFP-tagged BDNF in axons and dendrites of living corticalneurons by time-lapse imaging. In part of the experiments, the expression of BDNF tagged with cyan fluorescent protein (CFP was compared with that of nerve growth factor (NGF tagged with yellow fluorescent protein (YFP, to see whether fluorescent protein-tagged BDNF is expressed in a manner specific to this neurotrophin. Results We found that BDNF tagged with GFP or CFP was expressed in a punctated manner in dendrites and axons in about two-thirds of neurons into which plasmid cDNAs had been injected, while NGF tagged with GFP or YFP was diffusely expressed even in dendrites in about 70% of the plasmid-injected neurons. In neurons in which BDNF-GFP was expressed as vesicular puncta in axons, 59 and 23% of the puncta were moving rapidly in the anterograde and retrograde directions, respectively. On the other hand, 64% of BDNF-GFP puncta in dendrites did not move at all or fluttered back and forth within a short distance. The rest of the puncta in dendrites were moving relatively smoothly in either direction, but their mean velocity of transport, 0.47 ± 0.23 (SD μm/s, was slower than that of the moving puncta in axons (0.73 ± 0.26 μm/s. Conclusion The present results show that the pattern and velocity of the trafficking of fluorescence protein-tagged BDNF are different between axons and dendrites, and suggest that the anterograde transport in axons may be the dominant stream of BDNF to release sites.

Full Text Available Mutations in Leucine-Rich Repeat Kinase-2 (LRRK2 result in familial Parkinson’s disease and the G2019S mutation alone accounts for up to 30% in some ethnicities. Despite this, the function of LRRK2 is largely undetermined although evidence suggests roles in phosphorylation, protein interactions, autophagy and endocytosis. Emerging reports link loss of LRRK2 to altered synaptic transmission, but the effects of the G2019S mutation upon synaptic release in mammalian neurons are unknown. To assess wild type and mutant LRRK2 in established neuronal networks, we conducted immunocytochemical, electrophysiological and biochemical characterisation of >3 week old cortical cultures of LRRK2 knock-out, wild-type overexpressing and G2019S knock-in mice. Synaptic release and synapse numbers were grossly normal in LRRK2 knock-out cells, but discretely reduced glutamatergic activity and reduced synaptic protein levels were observed. Conversely, synapse density was modestly but significantly increased in wild-type LRRK2 overexpressing cultures although event frequency was not. In knock-in cultures, glutamate release was markedly elevated, in the absence of any change to synapse density; indicating that physiological levels of G2019S LRRK2 elevate probability of release. Several presynaptic regulatory proteins shown by others to interact with LRRK2 were expressed at normal levels in knock-in cultures; however, synapsin 1 phosphorylation was significantly reduced. Thus, perturbations to the presynaptic release machinery and elevated synaptic transmission are early neuronal effects of LRRK2 G2019S. Furthermore, the comparison of knock-in and overexpressing cultures suggests that one copy of the G2019S mutation has a more pronounced effect than an ~3-fold increase in LRRK2 protein. Mutant-induced increases in transmission may convey additional stressors to neuronal physiology that may eventually contribute to the pathogenesis of Parkinson’s disease.

Cyclin-dependent kinase 5 (Cdk5) activity is dependent on its association with 1 of 2 neuron-specific activators, p35 or p39. Cdk5 and its activators play an important role in brain development as well as higher functions like synaptic plasticity, learning, and memory. Reduction in p35 was reported in postmortem schizophrenia brain, in which reduced dendritic spine density was observed. Previous in vitro experiments have shown that Cdk5 is involved in dendritic spine formation, although in vivo evidence is limited. We examined dendritic spine formation in inducible-p35 conditional knockout (p35 cKO); p39 KO mice. When we deleted the p35 gene either during early postnatal days or at adult stage, we observed reduced spine densities of layer V neurons in the cerebral cortex and CA1 pyramidal neurons in the hippocampus. We further generated CA1-specific p35 conditional knockout (CA1-p35 cKO) mice and also CA1-p35 cKO; p39 KO mice in which have specific deletion of p35 in the CA1 region of hippocampus. We found a greater reduction in spine densities in CA1 pyramidal neurons in CA1-p35 cKO; p39 KO mice than in CA1-p35 cKO mice. These results indicate that dendritic spine formation and neuronal maintenance are dependent on Cdk5 activity.

Neocortical circuits are most sensitive to sensory experience during a critical period of early development. Previous studies implicate that brain-derived neurotrophic factor (BDNF) and GABAergic inhibition may control the timing of the critical period. By using an in vitro maturation model, we found that neurons at DIV (day in vitro) 7, around a period when functional synapses start to form and GABAergic inhibition emerges, displayed the most dynamic activation of extracellular signal-regulated kinase 1/2 (ERK1/2) and CREB by exogenous BDNF. The BDNF-stimulated transcriptional up-regulation of CREB target genes was also the highest in DIV 7 neurons. The basal level of ERK1/2 and CREB activity, as well as the expression of CREB target genes, increased along with maturation, and neurons at DIV 13 and 22 displayed less dynamic responses to BDNF. Furthermore, we found that the developmentally regulated GABAergic inhibition correlated with the decline of BDNF-mediated signaling during maturation. BDNF stimulation along with suppression of GABAergic inhibition enhanced the activation of ERK1/2-CREB signaling and gene transcription in mature neurons. Conversely, BDNF stimulation along with enhancement of GABAergic inhibition reduced the overall induction of intracellular signaling in younger neurons. We propose that the less dynamic molecular changes may play a certain role in the loss of plasticity during maturation.

BACKGROUND: Several studies have demonstrated that electroacupuncture by acupoint selection can inhibit cerebral corticalneuronal apoptosis following cerebral ischemia/reperfusion.OBJECTIVE: To validate the effects of electroacupuncture by acupoint selection on the expression level of corticalneuronal anti-apoptotic Bcl-2 protein and the apoptotic executive protein, caspase-3, in rat models of focal cerebral ischemia/reperfusion.DESIGN, TIME AND SETTING: This randomized grouping, neural cell and molecular biology animal experiment was performed at the Laboratory of Pharmacology of Traditional Chinese Medicine and the Laboratory Animal Center of Henan Institute of Traditional Chinese Medicine between November 2006 and May 2007.MATERIALS: Atotal of 40 healthy male adult Sprague-Dawley rats were randomly and evenly divided into four groups: sham-operated, model, electroacupuncture and non-acupoint control. G6895 electro-acupuncture instruments were purchased from Shanghai Huayi Instrument Factory, China. Caspase-3, Bcl-2 and Bax kits were provided by Wuhan Boster Bioengineering Co., Ltd., China.METHODS: Middle cerebral artery occlusion was induced in the model, electroacupuncture and non-acupoint groups. In the electroacupuncture group, the acupoints Jianyu (LI15), Waiguan (SJ5), Biguan (ST31), and Zusanli (ST36) were given electroacupuncture. In the non-acupoint control group, at each time point (immediately after ischemia and after reperfusion, or 2 hours after reperfusion), electroacupuncture was performed at the midpoints of Tianquan (PC2)-Quze (PC 3) line, Quze (PC 3)-Ximen (PC4) line, Zuwuli (LRlO)-Yinbao (LRg) line, and Xiguan (LR7)-Zhongdu (LR6) line. Electroacupuncture parameters were set with a continuous wave with a frequency of 10 Hz, wave width 0.6 ms, voltage 1.5-3.0 V, and a duration of 10 minutes. The sham-operated and model groups received only animal fixation without electroacupuncture procedure.MAIN OUTCOME MEASURES: Five rats were selected from

We compare the ability of two structurally different classes of epigenetic modulators, namely, histone deacetylase (HDAC) inhibitors containing either a hydroxamate or a mercaptoacetamide as the zinc binding group, to protect corticalneurons in culture from oxidative stress-induced death. This study reveals that some of the mercaptoacetamide-based HDAC inhibitors are fully protective, whereas the hydroxamates show toxicity at higher concentrations. Our present results appear to be consistent with the possibility that the mercaptoacetamide-based HDAC inhibitors interact with a different subset of the HDAC isozymes [less activity at HDAC1 and 2 correlates with less inhibitor toxicity], or alternatively, are interacting selectively with only the cytoplasmic HDACs that are crucial for protection from oxidative stress.

Ethanol produces changes in GABAA receptor trafficking and function that contribute to ethanol dependence symptomatology. Extrasynaptic γ-aminobutyric acid A receptors (GABAA-R) mediate inhibitory tonic current and are of particular interest because they are potentiated by physiologically relevant doses of ethanol. Here, we isolate GABAA α4δ receptors by western blotting in subsynaptic fractions to investigate protein kinase A (PKA) and protein kinase C (PKC) modulation of ethanol-induced receptor trafficking, while extrasynaptic receptor function is determined by measurement of tonic inhibition and responses evoked by 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP). Rat cerebral corticalneurons were grown for 18 days in vitro and exposed to ethanol and/or PKA/PKC modulators. Ethanol exposure (1 hour) did not alter GABAA α4 receptor abundance, but it increased tonic current amplitude, an effect that was prevented by inhibiting PKA, but not PKC. Direct activation of PKA, but not PKC, increased the abundance and tonic current of extrasynaptic α4δ receptors. In contrast, prolonged ethanol exposure (4 hours) reduced α4δ receptor abundance as well as tonic current, and this effect was also PKA dependent. Finally, PKC activation by ethanol or phorbol-12,13-dibutyrate (PdBu) had no effect on extrasynaptic α4δ subunit abundance or activity. We conclude that ethanol alters extrasynaptic α4δ receptor function and expression in corticalneurons in a PKA-dependent manner, but ethanol activation of PKC does not influence these receptors. These results could have clinical relevance for therapeutic strategies to restore normal GABAergic functioning for the treatment of alcohol use disorders.

To elucidate the utility of benzodiazepine receptor imaging for the detection of viable corticalneurons, dual-tracer autoradiography using iodine-125 iomazenil (IMZ) and iodine-123 N-isopropyl-4-iodoamphetamine (IMP) was performed in a model of reversible focal ischaemia during the acute and subacute phases. The right middle cerebral artery of anaesthetized rats was occluded for 60 min using an intraluminal filament and reperfused. In the acute phase study, {sup 125}I-IMZ (370 kBq) was injected via the femoral vein at 2 h after reperfusion, and {sup 123}I-IMP (37 MBq) was injected at 50 min post-injection. Rats were sacrificed 10 min after the injection of {sup 123}I-IMP. In the subacute phase study, the same procedure was performed at 5 days after reperfusion. In the acute phase, the IMP uptake was significantly decreased in almost all areas of the lesioned hemisphere, an exception being the cerebellum; however, the IMZ uptake was significantly decreased only in ischaemic cores. The discrepancy between IMZ and IMP uptake was observed in the lateral neocortex and the lateral caudate putamen (CPu), which were most frequently damaged in this ischaemic model. In the subacute phase, the IMZ uptake in lesioned rats was significantly decreased only in the parietal lobe and hippocampus, though the IMP uptake was decreased in many regions of lesioned hemispheres (the frontal, parietal cortex, CPu, hippocampus and thalamus). Histopathological findings indicated that both the IMP and the IMZ uptake was markedly decreased in necrotic areas. Although the IMP uptake was significantly decreased in the ischaemic areas, the IMZ uptake was maintained in these areas. These results suggest that benzodiazepine receptor imaging is superior to regional cerebral blood flow imaging for the detection of viable corticalneurons in both the acute and subacute phases of ischaemia. (orig.)

Objective. An increasing number of deaf individuals are being implanted with central auditory prostheses, but their performance has generally been poorer than for cochlear implant users. The goal of this study is to investigate stimulation strategies for improving hearing performance with a new auditory midbrain implant (AMI). Previous studies have shown that repeated electrical stimulation of a single site in each isofrequency lamina of the central nucleus of the inferior colliculus (ICC) causes strong suppressive effects in elicited responses within the primary auditory cortex (A1). Here we investigate if improved cortical activity can be achieved by co-activating ne